Polypeptide compositions comprising spacers

ABSTRACT

Disclosed herein are methods and compositions including antigen-binding polypeptides comprising a stalk region and a stalk extension region. In some cases, the antigen-binding compositions comprising the stalk extension region has increased expression on a cell surface and, in some cases, has increased antigen-binding efficiency. A subject antigen binding polypeptide can be a chimeric antigen receptor (CAR).

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of U.S. provisional PatentApplication No. 62/574,061; filed Oct. 18, 2017, which is herebyincorporated by reference in its entirety.

REFERENCE TO SEQUENCE LISTING

The present application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Oct. 11, 2018, is named50471-704_201_SL.txt and is 344,022 bytes in size.

BACKGROUND OF THE DISCLOSURE

Recombinant polypeptides such as chimeric polypeptides have been avaluable for research, diagnostic, manufacturing and therapeuticapplications. Indeed, adaptive T cell immunotherapy using chimericantigen receptors (CAR) and T-cell receptors (TCR) has been shown tosuccessfully direct killing of tumor cells. Modified effector cellsexpressing antigen binding polypeptides such as CARs are useful in thetreatment of diseases and disorders such as autoimmune disorders andcancers. In order to further develop this innovative technology it isvaluable to devise ways of increasing CAR expression on a cell surfaceand/or increasing antigen-binding efficiency.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

SUMMARY OF THE DISCLOSURE

Provided herein is a chimeric polypeptide comprising (i) anantigen-binding region, (ii) a transmembrane region, and (iii) a spacerregion connecting said trans-membrane region with said antigen bindingregion, wherein said spacer region comprises a stalk region(s)comprising at least one dimerization site, and a stalk extension region(s′-n), said stalk extension region comprising fewer dimerization sitesas compared to said stalk region.

Provided herein is a chimeric polypeptide comprising (i) anantigen-binding region, (ii) a transmembrane region, and (iii) a spacerregion connecting said trans-membrane region with said antigen bindingregion, wherein said spacer region comprises a stalk region which isfrom about 20 to about 60 amino acids in length and comprises at leastone dimerization site, and a stalk extension region comprising fromabout 1 to about 5 times the length of the stalk region as measured bynumber of amino acids.

Provided herein is a chimeric polypeptide comprising (i) anantigen-binding region, (ii) a transmembrane region, and (iii) a spacerregion connecting said trans-membrane region with the antigen bindingregion, wherein the spacer region comprises a stalk region designated as“s” and at least one stalk extension region, designated as “s′-n,”wherein n represents the number of units of s′ in the space region, andwherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20.

In some embodiments, at least one stalk extension region in the chimericpolypeptide has a sequence homologous to the stalk region except for thedimerization sites of the stalk region. In some embodiments, the spacerregion in the chimeric polypeptide is proximal to the membrane region.In some embodiments, the spacer region in the chimeric polypeptide isdistal to the membrane region. In some embodiments, the chimericpolypeptide further comprises an intracellular signaling domain. In someembodiments, the chimeric polypeptide does not comprise an intracellularsignaling domain.

In some embodiments, the stalk extension region of the chimericpolypeptide contains at least one fewer dimerization site as compared tothe stalk region. In one embodiment, the chimeric polypeptide hasimproved functional activity compared to an otherwise identicalantigen-binding polypeptide lacking the stalk extension region. In otherembodiments, the chimeric polypeptide has increased expression on a cellsurface compared to an otherwise identical polypeptide lacking the stalkextension region. In another embodiment, the stalk extension regionlacks a dimerization site. In yet another embodiment, each of the stalkextension regions is from about 20 to about 60 amino acids in length,wherein n is 1, 2, 3 or 4. In yet another embodiment, the stalkextension region has a sequence which has at least about 80% identity tothe stalk region. In some cases, at least one stalk extension region inthe chimeric polypeptide has a sequence comprising at least one lessdimerization site as compared to the stalk region. In some cases, eachof the stalk extension regions has a sequence which has at least about80% identity to the stalk region, wherein n is 2. In another case, eachof the stalk extension regions has a sequence which has at least about80% identity to the stalk region, wherein n is 3. In yet another case,each of the stalk extension region has a sequence which has at leastabout 80% identity to the stalk region, wherein n is 4. In yet anothercase, each of the stalk extension region has a sequence which has atleast about 80% identity to the stalk region, wherein n is at least 5.

In some cases, the stalk region of the chimeric polypeptide providedherein comprises a sequence with at least about 70%, 75%, 80%. 85%, 90%,95% or 99% identity to at least one of a CD8alpha hinge domain, a CD28hinge domain and a CTLA-4 hinge domain. In one embodiment, the stalkregion is a CD8 alpha hinge domain having a sequence as shown in SEQ IDNO: 1. In other embodiments, the stalk region is a CD28 hinge domainhaving a sequence as shown in SEQ ID NO: 7. In another embodiment, thestalk region is a CTLA-4 hinge domain having a sequence as shown in SEQID NO: 12.

In some embodiments, interchain dimerization of the chimeric polypeptideprovided herein is mediated by at least one disulfide bond betweencysteine residues. In some embodiments, the antigen binding region ofthe chimeric polypeptide binds an epitope on CD19. In some embodiments,the antigen binding region of the chimeric polypeptide binds an epitopeon CD33. In some embodiments, the antigen binding region of the chimericpolypeptide binds an epitope on at least one of CD19, BCMA, CD44,α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3,Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin,CD22, EGFR, GPC3, CSPG4, HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20,CD174, CD138, L1-CAM, FAP, c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2,CLL-1, Folate receptor α, Mucins, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA,TAG-72, EGFR, CD20, EGFRvIII, CD123 VEGF-R2, NY-ESO-1, Titin, MART-1,HPV, HBV, MAGE-A4, MAGE-A10, MAGE A3/A6, gp100, MAGE-A1, or PRAME.

In some embodiments, the chimeric polypeptide comprises a chimericantigen receptor (CAR). In some embodiments, the CAR further comprisesat least one costimulatory signaling domain. In some embodiments, the atleast one costimulatory signaling domain comprises a signaling domainfrom CD27, CD28, 4-1BB, ICOS, OX40, DAP10, DAP12, CD134, CD3-zeta orfragment or combination thereof. In some embodiments, the at least onecostimulatory signaling domain comprises a signaling domain from 4-1BB,CD28 or a combination thereof. In some embodiments, the CAR furthercomprises a CD28 costimulatory signaling domain and CD3-zeta. In someembodiments, the CAR further comprises a CD28 costimulatory signalingdomain. In some embodiments, the intracellular cell signaling domaininteracts with a T cell, a Natural Killer (NK) cell, a cytotoxic Tlymphocyte (CTL), or a regulatory T cell.

In some embodiments, the chimeric polypeptide comprises an engineeredT-cell receptor (TCR). In some embodiments, the engineered TCR is an αβTCR. In some embodiments, the engineered TCR is a γδ TCR. In someembodiments, the antigen binding region of the engineered TCR binds anepitope on at least one of CD19, BCMA, CD44, α-Folate receptor, CAIX,CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2,GD3, IL-13R-a2, KDR, EDB-F, mesothelin, CD22, EGFR, GPC3, CSPG4,HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP,c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2, CLL-1, Folate receptor α,Mucins, MUC-1, MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20,EGFRvIII, CD123, VEGF-R2, NY-ESO-1, Titin, MART-1, HPV, HBV, MAGE-A4,MAGE-A10, MAGE A3/A6, gp100, MAGE-A1, or PRAME. In some embodiments, theantigen binding region of the engineered TCR binds an epitope on atleast one of NY-ESO-1, Titin, MART-1, HPV, HBV, MAGE-A4, MAGE-A10, MAGEA3/A6, gp100, MAGE-A1, or PRAME.

Also provided is a polynucleotide encoding the chimeric polypeptide asdescribed herein. Provided herein is an expression vector comprising thepolynucleotide. In some embodiments, the vector is a lentivirus vector,a retroviral vector or a non-viral vector. In some embodiments, thevector is a non-viral vector which is a sleeping beauty vector.

Further provided is an engineered cell comprising the expression vectorcomprising the polypeptide encoding the chimeric polypeptide asdescribed herein. In some cases, the engineered cell further comprises aSleeping Beauty transposase. In some cases, the Sleeping Beautytransposase is SB11, SB100X or SB110. In some cases, the engineered cellis an animal cell. In some cases, the animal cell is a human cell. Insome cases, the human cell is a T cell or NK cell. In some cases, theengineered cell as described herein further expresses a polypeptidecomprising an intracellular signaling domain.

Provided herein is a chimeric antigen receptor (CAR) comprising anantigen binding region, a transmembrane region a stalk region and astalk extension region, wherein the stalk extension region is homologousto the stalk region and comprises at least one amino acid substitutionrelative to the stalk region. In some embodiments, the stalk region iscapable of dimerizing with a homologous stalk region of a second CAR. Insome embodiments, the stalk extension region is not capable ofdimerizing. In some embodiments, the stalk region is connected to thetransmembrane region. In some embodiments, the antigen binding regioncomprises a scFv. In some embodiments, the scFv binds an epitope onCD19, BCMA, CD44, α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2,EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR,EDB-F, mesothelin, CD22, GPC3, CSPG4, HER1/HER3, HER2, CD44v6,CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP, c-MET, PSCA, CS1, CD38,IL-11Rα, EphA2, CLL-1, EGFR, Folate receptor α, Mucins, MUC-1, MUC-16,MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII, CD123 or VEGF-R2.

In some embodiments, the stalk extension region of the CAR is designatedas s′-n, wherein n comprises two or more, thereby comprising a firststalk extension region and a second stalk extension region. In someembodiments, the first stalk extension region is homologous to thesecond stalk extension region. In some embodiments, the first stalkextension region comprises at least one amino acid residue substitutioncompared to the stalk region. In some embodiments, the first stalkextension region is not capable of dimerizing to a stalk region of aCAR. In some embodiments, the second stalk extension region comprises atleast one amino acid residue substitution compared to the stalk region.In some embodiments, the second stalk extension region is not capable ofdimerizing to another stalk extension region. In some embodiments, theCAR further comprises a third stalk extension region. In someembodiments, the CAR further comprises a fourth stalk extension region.In some embodiments, the CAR comprises five or more stalk extensionregion. In some embodiments, at least one stalk extension region in theCAR is not capable of forming a disulfide bond. In some embodiments, thestalk region comprises a sequence with at least about 70%, 75%, 80%.85%, 90%, 95% or 99% identity to at least one of a CD8alpha hingedomain, a CD28 hinge domain and a CTLA-4 hinge domain. In someembodiments, the stalk region is a CD8alpha hinge domain, a CD28 hingedomain or a CTLA-4 hinge domain.

In some cases, a T cell or NK cell expresses the CAR as describedherein. In some cases, the CAR comprises the sequence shown as SEQ IDNo. 53-68, or a variant thereof which has at least 80% sequence identitybut retains antigen binding capacity.

Provided herein is a nucleic acid sequence encoding the CAR as presentlydescribed. In some cases, the nucleic acid sequence comprises thesequence shown as SEQ ID No 147-162 or a variant thereof having at least70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity.

Provided herein is a vector comprising the nucleic acid sequenceencoding the CAR as presently described. In some embodiments, the vectoris a lentivirus vector, a retroviral vector, a Sleeping Beautytransposon or a non-viral vector.

Provided herein is a method for making a T cell or NK cell, wherein themethod comprises the step of introducing the nucleic acid sequenceencoding the CAR as presently described into the T cell or NK cell. Insome embodiments, the T cell is modified at a point-of-care site andadministered to a subject in need thereof, without undergoingpropagation and activation. In some embodiments, the T cell isstimulated for at least 0, 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,24, 26, 28, 30 or more days. In some embodiments, the T cell isstimulated for at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 ormore days. In some embodiments, the T cell is stimulated for at least 7,14, 21, 28, 35, 42, 49, 56, 63 or more days.

Provided herein is a pharmaceutical composition which comprises at leastone of: a vector comprising the nucleic acid sequence encoding the CARas presently described; and a T cell or a NK cell expressing the CAR aspresently described; and a pharmaceutically acceptable carrier, diluentor excipient.

Provided herein is a population of cells comprising the CAR as presentlydescribed.

Provided herein is a method for stimulating a T cell-mediated immuneresponse in a subject, comprising contacting the subject with aneffective amount of a cell genetically modified to express the CAR aspresently described.

Provided herein is a method of increasing expression of a chimericantigen receptor (CAR) on a cell surface comprising engineering anucleic acid encoding the CAR to comprise a stalk extension domain,thereby generating an engineered CAR.

Provided herein is a method of increasing expansion of an engineered Tcell expressing a chimeric polypeptide comprising engineering a nucleicacid encoding the chimeric polypeptide to comprise a stalk extensiondomain, thereby generating an engineered T cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The features of the present disclosure are set forth with particularityin the appended claims. A better understanding of the features andadvantages of the present disclosure will be obtained by reference tothe following detailed description that sets forth illustrativeembodiments, in which the principles of the disclosure are utilized, andthe accompanying drawings of which:

FIG. 1A depicts diagrams of polypeptides with spacers that incorporate astalk and varying numbers of stalk extension regions (s′-1, s′-2, s′-3).FIG. 1B depicts diagrams of polypeptides with spacers that incorporate astalk and varying numbers of stalk extension regions (s′-1, s′-2, s′-3).The diagrams also depict exemplary dimerization sites.

FIG. 2A depicts diagrams of exemplary polypeptides such as chimericantigen receptors, with various spacer lengths. FIG. 2B depicts diagramsof exemplary polypeptides such as chimeric antigen receptors, withvarious spacer lengths. The illustration shows chimeric antigenreceptors with spacers that incorporate a stalk and one, two or threestalk extension regions.

FIGS. 3A-3B depict exemplary data showing expansion of CD19-CAR-T cellsexpressing CARs of varying spacer lengths in ex vivo culture. FIG. 3Ashows that Sleeping Beauty modified CD19-CAR-T cells were proliferatedex vivo in presence of Activating and Propagating Cells (AaPC)expressing CD19 antigen on the surface. CAR-T cells expressing CARs withspacers incorporating one, two or three stalk extension regions (CD8-2×,CD8-3× and CD8-4×) showed similar proliferative potential compared toCAR with a spacer incorporating a CD8 stalk. T cells expressing CARswith spacers incorporating stalk extension regions CD8-3×v2 containingamino acid substitutions in stalk extension region compared to CD8-3×failed to expand ex vivo. FIG. 3B demonstrates higher expression (MFI)of CD19 CAR with spacers incorporating two stalk extension regions(CD8-3×) as compared to CD19 CAR with spacers incorporating a CD8α hingestalk (CD8-1×).

FIG. 4 shows expression of CD19-specific CARs of varying spacer lengthsin T cells as measured by western blot analysis.

FIGS. 5A-5B show that T cells expressing CD19-CARs with varying spacerlengths are capable of exerting specific cytotoxicity effects againsttarget cells expressing CD19. FIG. 5A shows cytotoxic activity of Tcells expressing CD19-CAR with varying spacer lengths against a K562cell line engineered to express CD19 (K562/CD19) antigen at differenteffector to target cell (E:T) ratios. T cells expressing CARs withspacers incorporating one, two or three stalk extension regions (CD8-2×,CD8-3× and CD8-4×) exerted similar cytotoxicity at 10:1 E:T ratio andimproved cytotoxicity at lower E:T ratios of K562/CD19 cell linecompared to T cells expressing CARs incorporating a spacer with just aCD8α hinge stalk (CD8-1×). FIG. 5B shows cytotoxic response of T cellsexpressing CD19-CAR with varying spacer lengths at E:T ratio of 10:1towards CD19 negative K562 or EL4 cell lines as well as CD19 positiveK562/CD19 cell line. T cells expressing CD19-CARs with spacersincorporating one, two or three stalk extension regions (CD8-2×, CD8-3×and CD8-4×) exerted similar cytotoxicity effects against the K562/CD19cell line compared to T cells expressing CAR with a CD8α hinge stalk(CD8-1×). However, T cells expressing CARs with extended stalk lengthregions (CD8-2×, CD8-3× and CD8-4×) showed lower non-specificcytotoxicity of CD19 negative K562 and EL4 cell lines.

FIGS. 6A-6C show the ability of T cells expressing CD19-CARs withvarying spacer lengths to produce cytokines in response to CD19 antigenexpressing cells. Levels of cytokines released in response to CD19negative cell lines (K562 and EL4) were undetectable or very lowdemonstrating specificity of CD19 CAR-T cells towards CD19 antigen. FIG.6A demonstrates that T cells expressing CD19-specific CARs with spacersincorporating one, two or three stalk extension regions (CD8-2×, CD8-3×and CD8-4×) demonstrated an enhanced release of IFNγ cytokine comparedto T cells expressing CARs with CD8α hinge stalk (CD8-1×) when culturedwith CD19 positive K562/CD19 cell line at 10:1 E:T ratio. FIG. 6Bdemonstrates that T cells expressing CD19-specific CARs with spacersincorporating one, two or three stalk extension regions (CD8-2×, CD8-3×and CD8-4×) demonstrated an enhanced release of TNF cytokine compared toT cells expressing CARs with CD8α hinge stalk and no stalk extensionregions (CD8-1×) when cultured with CD19 positive K562/CD19 cell line at10:1 E:T ratio. FIG. 6C demonstrates that T cells expressingCD19-specific CARs with spacers incorporating one, two or three stalkextension regions (CD8-2×, CD8-3× and CD8-4×) demonstrated an enhancedrelease of Granzyme B cytokine compared to T cells expressing CARs withCD8α hinge stalk (CD8-1×) when cultured with CD19 positive K562/CD19cell line at 10:1 E:T ratio.

FIG. 7A depicts expression data for ROR-1 CARs with varying spacerlengths after successive rounds of stimulation on aAPC. FIG. 7B depictsexpression data for ROR-1 CARs with varying spacer lengths aftersuccessive rounds of stimulation on aAPC. As seen from the data, theincorporation of stalk extension regions resulted in improvedexpression.

FIG. 8A depicts exemplary data from functional activity assays tomeasure degranulation of T cells expressing ROR-1 CAR with varyingspacer lengths, as measured by CD107a expression and IFNγ release. FIG.8B depicts exemplary data from functional activity assays to measuredegranulation of T cells expressing CAR with varying spacer lengths, asmeasured by CD107a expression and IFNγ release.

FIG. 9 shows expression of CD33-specific CAR with varying spacer lengthson surface of T cells. Human PBMCs were electroporated with SleepingBeauty transposons encoding for CD33-specific CAR with spacersincorporating one, two or three stalk extension regions (CD8-2×, CD8-3×and CD8-4×) and co-cultured with AaPC expressing CD33 antigen for exvivo proliferation of CAR-T cells. Expression of CD33-specific CAR wasmeasured by flow cytometry using CD33-Fc protein. CD33-specific CARswith spacers incorporating two or three stalk extension regions (CD8-3×and CD8-4×) exhibited improved expression and CAR-T cell growth at Day 7when compared with CD33-specific CAR with CD8α hinge stalk (CD8-1×).

FIG. 10A depicts diagrams of engineered T cell receptors (TCR) withspacers that incorporate a stalk (s) and varying numbers of stalkextension regions (s′-1, s′-2). FIG. 10B depicts diagrams of engineeredT cell receptors (TCR) with spacers that incorporate a stalk (s) andvarying numbers of stalk extension regions (s′-1, s′-2). The diagramsalso depict exemplary disulfide bond mediated dimerization sites.

FIG. 11A depicts expression data for MR1-1 and huMR1-1 CARs specific forEGFRvIII with varying spacer lengths after a round of stimulation onaAPC. FIG. 11B depicts expression data for MR1-1 and huMR1-1 CARsspecific for EGFRvIII with varying spacer lengths after three rounds ofstimulation on aAPC. As seen from the data, the incorporation of stalkextension regions resulted in improved expression. FIG. 11C-D depictstotal number of CAR⁺ T cells and fold expansion of huMR1-1 CAR⁺ T cellswith varying spacer lengths after consecutive rounds of AaPCstimulations. FIG. 11E depicts the depicts exemplary data measuringcytotoxicity activity of T cells expressing huMR1-1 CAR⁺ T cells withvarying spacer lengths, as measured by Europium release assay.

DETAILED DESCRIPTION OF THE DISCLOSURE

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. It is to be understoodthat the detailed description are exemplary and explanatory only and arenot restrictive of any subject matter claimed. In this application, theuse of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, use of the term“including” as well as other forms, such as “include”, “includes,” and“included,” is not limiting.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features may be described in the context of a singleembodiment, the features may also be provided separately or in anysuitable combination. Conversely, although the present disclosure may bedescribed herein in the context of separate embodiments for clarity, itmay also be implemented in a single embodiment.

Reference in the specification to “some embodiments”, “an embodiment”,“one embodiment” or “other embodiments” means that a particular feature,structure, or characteristic described in connection with theembodiments is included in at least some embodiments, but notnecessarily all embodiments described herein.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps. It is contemplated that any embodimentdiscussed in this specification can be implemented with respect to anymethod or composition described herein, and vice versa. Furthermore,compositions described herein can be used to achieve methods describedherein.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 μL” means “about 5 μL” and also “5 μL.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

By “isolated” or its grammatical equivalent is meant the removal of anucleic acid from its natural environment. By “purified” or itsgrammatical equivalent is meant that a given nucleic acid, whether onethat has been removed from nature (including genomic DNA and mRNA) orsynthesized (including cDNA) and/or amplified under laboratoryconditions, has been increased in purity, wherein “purity” is a relativeterm, not “absolute purity.” It is to be understood, however, thatnucleic acids and proteins may be formulated with diluents or adjuvantsand still for practical purposes be isolated. For example, nucleic acidstypically are mixed with an acceptable carrier or diluent when used forintroduction into cells.

“Polynucleotide” or “oligonucleotide” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double and single stranded DNA,triplex DNA, as well as double and single stranded RNA. It also includesmodified, for example, by methylation and/or by capping, and unmodifiedforms of the polynucleotide. The term is also meant to include moleculesthat include non-naturally occurring or synthetic nucleotides as well asnucleotide analogs.

“Polypeptide” is used interchangeably with the terms “polypeptides,”“peptide(s),” and “protein(s)”, and refers to a polymer of amino acidresidues. A “mature protein” is a protein which is full-length andwhich, optionally, includes glycosylation or other modifications typicalfor the protein in a given cellular environment.

Nucleic acids and/or nucleic acid sequences are “homologous” when theyare derived, naturally or artificially, from a common ancestral nucleicacid or nucleic acid sequence. Proteins and/or protein sequences arehomologous when their encoding DNAs are derived, naturally orartificially, from a common ancestral nucleic acid or nucleic acidsequence. The homologous molecules can be termed homologs. For example,any naturally occurring proteins, as described herein, can be modifiedby any available mutagenesis method. When expressed, this mutagenizednucleic acid encodes a polypeptide that is homologous to the proteinencoded by the original nucleic acid. Homology is generally inferredfrom sequence identity between two or more nucleic acids or proteins (orsequences thereof). The precise percentage of identity between sequencesthat is useful in establishing homology varies with the nucleic acid andprotein at issue, but as little as 25% sequence identity is routinelyused to establish homology. Higher levels of sequence identity, e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can also be usedto establish homology.

The terms “identical” or “sequence identity” in the context of twonucleic acid sequences or amino acid sequences of polypeptides refers tothe residues in the two sequences which are the same when aligned formaximum correspondence over a specified comparison window.

In one class of embodiments, the polypeptides herein are at least 80%,85%, 90%, 98% 99% or 100% identical to a reference polypeptide, or afragment thereof, e.g., as measured by BLASTP (or CLUSTAL, or any otheravailable alignment software) using default parameters. Similarly,nucleic acids can also be described with reference to a starting nucleicacid, e.g., they can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99% or100% identical to a reference nucleic acid or a fragment thereof, e.g.,as measured by BLASTN (or CLUSTAL, or any other available alignmentsoftware) using default parameters. When one molecule is said to havecertain percentage of sequence identity with a larger molecule, it meansthat when the two molecules are optimally aligned, said percentage ofresidues in the smaller molecule finds a match residue in the largermolecule in accordance with the order by which the two molecules areoptimally aligned.

Proteins disclosed herein (including functional portions and functionalvariants thereof) can comprise synthetic amino acids in place of one ormore naturally-occurring amino acids. Such synthetic amino acids areknown in the art, and include, for example, aminocyclohexane carboxylicacid, norleucine, α-amino n-decanoic acid, homoserine,S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline,4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine,4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine,phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine,indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid, aminomalonic acid, aminomalonic acid monoamide,N′-benzyl-N′-methyl-lysine, N′,N′-dibenzyl-lysine, 6-hydroxylysine,ornithine, α-aminocyclopentane carboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptane carboxylic acid,α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid,α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

“Transposon” or “transposable element” (TE) is a vector DNA sequencethat can change its position within the genome, sometimes creating orreversing mutations and altering the cell's genome size. Transpositionoften results in duplication of the TE. Class I TEs are copied in twostages: first they are transcribed from DNA to RNA, and the RNA producedis then reverse transcribed to DNA. This copied DNA is then inserted ata new position into the genome. The reverse transcription step iscatalyzed by a reverse transcriptase, which may be encoded by the TEitself. The characteristics of retrotransposons are similar toretroviruses, such as HIV. The cut-and-paste transposition mechanism ofclass II TEs does not involve an RNA intermediate. The transpositionsare catalyzed by several transposase enzymes. Some transposasesnon-specifically bind to any target site in DNA, whereas others bind tospecific DNA sequence targets. The transposase makes a staggered cut atthe target site resulting in single-strand 5′ or 3′ DNA overhangs(sticky ends). This step cuts out the DNA transposon, which is thenligated into a new target site; this process involves activity of a DNApolymerase that fills in gaps and of a DNA ligase that closes thesugar-phosphate backbone. This results in duplication of the targetsite. The insertion sites of DNA transposons may be identified by shortdirect repeats which may be created by the staggered cut in the targetDNA and filling in by DNA polymerase, followed by a series of invertedrepeats important for the TE excision by transposase. Cut-and-paste TEsmay be duplicated if their transposition takes place during S phase ofthe cell cycle when a donor site has already been replicated, but atarget site has not yet been replicated. Transposition can be classifiedas either “autonomous” or “non-autonomous” in both Class I and Class IITEs. Autonomous TEs can move by themselves while non-autonomous TEsrequire the presence of another TE to move. This is often becausenon-autonomous TEs lack transposase (for class II) or reversetranscriptase (for class I).

“Transposase” refers an enzyme that binds to the end of a transposon andcatalyzes the movement of the transposon to another part of the genomeby a cut and paste mechanism or a replicative transposition mechanism.In some embodiments, the transposase's catalytic activity can beutilized to move gene(s) from a vector to the genome.

In some instances, polynucleotides encoding gene-switch polypeptides forexpressing CARs and/or TCRs described herein can also be introduced intoT cells using non-viral based delivery systems, such as the “SleepingBeauty (SB) Transposon System,” which refers a synthetic DNA transposonsystem for introducing DNA sequences into the chromosomes ofvertebrates. Some exemplary embodiments of the system are described, forexample, in U.S. Pat. Nos. 6,489,458; 8,227,432; 9,228,180 andWO/2016/145146. The Sleeping Beauty transposon system is composed of aSleeping Beauty (SB) transposase and a SB transposon. In embodiments,the Sleeping Beauty transposon system can include the SB11 transposonsystem, the SB100X transposon system, or the SB110 transposon system.

The nucleic acid sequences and vectors disclosed or contemplated hereincan be introduced into a cell by “transfection,” “transformation,” or“transduction.” “Transfection,” “transformation,” or “transduction,” asused herein, refer to the introduction of one or more exogenouspolynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art and include, forexample, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J.(ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression Protocols, Humana Press (1991)); DEAE-dextran;electroporation; cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors canbe introduced into host cells, after growth of infectious particles insuitable packaging cells, many of which are commercially available.

“Promoter” refers to a region of a polynucleotide that initiatestranscription of a coding sequence. Promoters are located near thetranscription start sites of genes, on the same strand and upstream onthe DNA (towards the 5′ region of the sense strand). Some promoters areconstitutive as they are active in all circumstances in the cell, whileothers are regulated becoming active in response to specific stimuli,e.g., an inducible promoter.

The term “promoter activity” refers to the extent of expression ofnucleotide sequence that is operably linked to the promoter whoseactivity is being measured. Promoter activity may be measured directlyby determining the amount of RNA transcript produced, for example byNorthern blot analysis or indirectly by determining the amount ofproduct coded for by the linked nucleic acid sequence, such as areporter nucleic acid sequence linked to the promoter.

“Inducible promoter” as used herein refers to a promoter which isinduced into activity by the presence or absence of transcriptionalregulators, e.g., biotic or abiotic factors. Inducible promoters areuseful because the expression of genes operably linked to them can beturned on or off at certain stages of development of an organism or in aparticular tissue. Examples of inducible promoters are alcohol-regulatedpromoters, tetracycline-regulated promoters, steroid-regulatedpromoters, metal-regulated promoters, pathogenesis-regulated promoters,temperature-regulated promoters and light-regulated promoters. In oneembodiment, the inducible promoter is part of a genetic switch.

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences. The term “Igenhancers” refers to enhancer elements derived from enhancer regionsmapped within the immunoglobulin (Ig) locus (such enhancers include forexample, the heavy chain (mu) 5′ enhancers, light chain (kappa) 5′enhancers, kappa and mu intronic enhancers, and 3′ enhancers (seegenerally Paul W. E. (ed), Fundamental Immunology, 3rd Edition, RavenPress, New York (1993), pages 353-363; and U.S. Pat. No. 5,885,827).

An “expression vector” or “vector” is any genetic element, e.g., aplasmid, chromosome, virus, transposon, behaving either as an autonomousunit of polynucleotide replication within a cell. (i.e. capable ofreplication under its own control) or being rendered capable ofreplication by insertion into a host cell chromosome, having attached toit another polynucleotide segment, so as to bring about the replicationand/or expression of the attached segment. Suitable vectors include, butare not limited to, plasmids, transposons, bacteriophages and cosmids.Vectors may contain polynucleotide sequences which are necessary toeffect ligation or insertion of the vector into a desired host cell andto effect the expression of the attached segment. Such sequences differdepending on the host organism; they include promoter sequences toeffect transcription, enhancer sequences to increase transcription,ribosomal binding site sequences and transcription and translationtermination sequences. Alternatively, expression vectors can be capableof directly expressing nucleic acid sequence products encoded thereinwithout ligation or integration of the vector into host cell DNAsequences.

Vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allows cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/08796 and WO 1994/28143; Wigler et al., Proc. Natl. Acad. Sci. USA,77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527(1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981);Colberre-Garapin et al., J. Mol. Biol., 150:1(1981); Santerre et al.,Gene, 30: 147 (1984); Kent et al., Science, 237: 901-903 (1987); Wigleret al., Cell, 11: 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad.Sci. USA, 48: 2026 (1962); Lowy et al., Cell, 22: 817 (1980); and U.S.Pat. Nos. 5,122,464 and 5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al., Gene Therapy,11:1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

“Antibody” as used herein refers to monoclonal or polyclonal antibodies.The term “monoclonal antibodies,” as used herein, refers to antibodiesthat are produced by a single clone of B-cells and bind to the sameepitope. In contrast, “polyclonal antibodies” refer to a population ofantibodies that are produced by different B-cells and bind to differentepitopes of the same antigen. A whole antibody typically consists offour polypeptides: two identical copies of a heavy (H) chain polypeptideand two identical copies of a light (L) chain polypeptide. Each of theheavy chains contains one N-terminal variable (VH) region and threeC-terminal constant (CH1, CH2 and CH3) regions, and each light chaincontains one N-terminal variable (VL) region and one C-terminal constant(CL) region. The variable regions of each pair of light and heavy chainsform the antigen binding site of an antibody. The VH and VL regions havea similar general structure, with each region comprising four frameworkregions, whose sequences are relatively conserved. The framework regionsare connected by three complementarity determining regions (CDRs). Thethree CDRs, known as CDR1, CDR2, and CDR3, form the “hypervariableregion” of an antibody, which is responsible for antigen binding.

The terms “fragment of an antibody,” “antibody fragment,” “functionalfragment of an antibody,” and “antigen-binding portion” are usedinterchangeably herein to mean one or more fragments or portions of anantibody that retain the ability to specifically bind to an antigen(see, generally, Holliger et al., Nat. Biotech., 23(9):1126-1129(2005)). The antibody fragment desirably comprises, for example, one ormore CDRs, the variable region (or portions thereof), the constantregion (or portions thereof), or combinations thereof. Examples ofantibody fragments include, but are not limited to, (i) a Fab fragment,which is a monovalent fragment consisting of the VL, VH, CL, and CH1domains; (ii) a F(ab′)2 fragment, which is a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the stalkregion; (iii) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody; (iv) a single chain Fv (scFv), which is amonovalent molecule consisting of the two domains of the Fv fragment(i.e., VL and VH) joined by a synthetic linker which enables the twodomains to be synthesized as a single polypeptide chain (see, e.g., Birdet al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad.Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol.,16: 778 (1998)) and (v) a diabody, which is a dimer of polypeptidechains, wherein each polypeptide chain comprises a VH connected to a VLby a peptide linker that is too short to allow pairing between the VHand VL on the same polypeptide chain, thereby driving the pairingbetween the complementary domains on different VH-VL polypeptide chainsto generate a dimeric molecule having two functional antigen bindingsites. Antibody fragments are known in the art and are described in moredetail in, e.g., U.S. Patent Application Publication 2009/0093024 A1.

The term “functional portion,” when used in reference to a CAR, refersto any part or fragment of the CAR described herein, which part orfragment retains the biological activity of the CAR of which it is apart (the parent CAR). In reference to a nucleic acid sequence encodingthe parent CAR, a nucleic acid sequence encoding a functional portion ofthe CAR can encode a protein comprising, for example, about 10%, 25%,30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CAR.

The term “functional variant,” as used herein, refers to a polypeptide,or a protein having substantial or significant sequence identity orsimilarity to the reference polypeptide, and retains the biologicalactivity of the reference polypeptide of which it is a variant.Functional variants encompass, for example, those variants of the CARdescribed herein (the parent CAR) that retain the ability to recognizetarget cells to a similar extent, the same extent, or to a higherextent, as the parent CAR. In reference to a nucleic acid sequenceencoding the parent CAR, a nucleic acid sequence encoding a functionalvariant of the CAR can be for example, about 10% identical, about 25%identical, about 30% identical, about 50% identical, about 65%identical, about 80% identical, about 90% identical, about 95%identical, or about 99% identical to the nucleic acid sequence encodingthe parent CAR.

“Proliferative disease” as referred to herein means a unifying conceptthat excessive proliferation of cells and turnover of cellular matrixcontribute significantly to the pathogenesis of several diseases,including cancer is presented.

“Administering” is referred to herein as providing the compositionsdescribed herein to a patient. By way of example and not limitation,composition administration, e.g., injection, may be performed byintravenous (i.v.) injection, sub-cutaneous (s.c.) injection,intradermal (i.d.) injection, intraperitoneal (i.p.) injection, orintramuscular (i.m.) injection. One or more such routes may be employed.Parenteral administration can be, for example, by bolus injection or bygradual perfusion over time. Alternatively, or concurrently,administration may be by the oral route. Additionally, administrationmay also be by surgical deposition of a bolus or pellet of cells, orpositioning of a medical device.

Modified or engineered cell compositions described herein may compriseshost cells expressing one or more nucleic acid sequences describedherein, or a vector comprising one or more nucleic acid sequencesdescribed herein, in an amount that is effective to treat or preventproliferative disorders. As used herein, the terms “treatment,”“treating,” and the like refer to obtaining a desired pharmacologicand/or physiologic effect. In embodiments, the effect is therapeutic,i.e., the effect partially or completely cures a disease and/or adversesymptom attributable to the disease. To this end, the inventive methodcomprises administering an “amount” of the composition comprising thehost cells expressing the inventive nucleic acid sequence, or a vectorcomprising the inventive nucleic acid sequences.

An “amount” or “dose” refers to an amount effective, at dosages and forperiods of time necessary, to achieve a desired therapeutic result. Theamount can vary according to factors such as the disease state, age,sex, and weight of the individual, and the ability of the inventivenucleic acid sequences to elicit a desired response in the individual.

Alternatively, the pharmacologic and/or physiologic effect can be“prophylactic,” i.e., the effect completely or partially prevents adisease or symptom thereof.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredprophylactic result (e.g., prevention of disease onset).

As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)”mean any mammal. In some embodiments, the mammal is a human. In someembodiments, the mammal is a non-human. None of the terms require or arelimited to situations characterized by the supervision (e.g. constant orintermittent) of a health care worker (e.g. a doctor, a registerednurse, a nurse practitioner, a physician's assistant, an orderly or ahospice worker).

Spacers

Described herein are spacers that connect two regions of a polypeptideconstruct described herein. For instance, spacers described herein canconnect a transmembrane region of a polypeptide to an antigen or ligandbinding region of a polypeptide. In some cases, the polypeptide can be achimeric polypeptide. A chimeric polypeptide or chimeric protein asdescribed herein includes polypeptides or proteins created by joining oftwo or more genes or portions or derivatives thereof, that originallycoded for separate proteins. Exemplary depictions of various spacers canbe found at FIGS. 1 and 2 . In some embodiments, the spacer extends thedistance between different domains of a chimeric polypeptide resultingin improved expression or functional activity of the polypeptidecompared to an otherwise identical polypeptide lacking the spacer. Insome instances, a spacer comprises any polypeptide that functions tolink the transmembrane region to, either the extracellular region or,the cytoplasmic region in the chimeric polypeptide. In some embodiments,the spacer is flexible enough to allow the antigen or ligand-bindingregion to align in different orientations to facilitate antigen orligand receptor recognition. In other embodiments, the spacer extendsthe distance between different domains of a chimeric polypeptideresulting in improved expansion or propagation of the cell(s) thatexpresses the chimeric polypeptide compared to cell(s) expressing anotherwise identical polypeptide lacking the spacer.

The spacer can comprise a stalk region and stalk extension region(s). Inone embodiment, a spacer can include a single stalk region. In anotherembodiment, a spacer can comprise a stalk region (designated as “s”) andstalk extension region(s), which is herein designated as “s′-n.” Forexample, a spacer can comprise one (1) stalk region and s′-n, wherein ncan be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20. In further embodiments, the stalk region can be linked tostalk extension region s′-n via a linker. A linker as described hereincan include for instance, a GSG linker (SEQ ID NO: 9 and SEQ ID NO:115), SGSG linker (SEQ ID NO: 10 and SEQ ID NO: 116), (G4S)3 linker (SEQID NO: 11 and SEQ ID NO: 117), (G4S)4 linker (SEQ ID NO: 255) and/or aWhitlow linker (SEQ ID NO: 8 and SEQ ID NO: 114). In certain cases, apeptide linker of any length or size to link a stalk region and stalkextension region(s). For example, in some embodiments, a peptide linkeris sized to maintain a desired or optimal distance between the stalkregion and stalk extension region. In some embodiments are differentsize G4S linkers (SEQ ID NO: 256) (G4S)n, wherein n=0, 1, 2, 3, 4, 5(SEQ ID NO: 257).

In some embodiments, the stalk region can be from about 20 to about 300amino acids in length and comprises at least one dimerization site, anda stalk extension region can comprise from about 1 to about 10 times thelength of the stalk region as measured by number of amino acids.

In some cases, a stalk region can be about 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 orgreater amino acids in length. In other cases, the stalk region can beabout: 100, 125, 150, 175, 200, 225, 250, 275 or 300 amino acids inlength. In some cases, a stalk region can be less than 20 amino acids inlength.

In some cases, a stalk extension region can comprise from about 1 toabout 10 times the length of the stalk region as measured by number ofamino acids. For example, a stalk extension region can comprise about 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 times the length of the stalk region asmeasured by number of amino acids. In some cases, a stalk extensionregion can comprise greater than 10 times the length of the stalk regionas measured by number of amino acids. In some examples, a stalkextension region can comprise up to 2 times the length of the stalkregion as measured by number of amino acids but comprise fewerdimerization sites than the stalk region.

A stalk extension region of a subject antigen-binding polypeptide cancontain at least one fewer dimerization site as compared to a stalkregion. For example, if a stalk region comprises two dimerization sites,a stalk extension region can comprise one or zero dimerization sites. Asanother example, if a stalk region comprises one dimerization site, astalk extension region can comprise zero dimerization sites. In someexamples, a stalk extension region lacks a dimerization site. In someexamples, a stalk extension region can comprise up to 2 times the lengthof the stalk region as measured by number of amino acids but comprise nodimerization sites. In some examples, a stalk extension region cancomprise up to 3 times the length of the stalk region as measured bynumber of amino acids but comprise no dimerization sites. In someexamples, a stalk extension region can comprise up to 4 times the lengthof the stalk region as measured by number of amino acids but comprisezero dimerization sites. In some cases, one or more dimerization site(s)can be membrane proximal. In other cases, one or more dimerizationsite(s) can be membrane distal.

Each of the stalk extension regions can, in some examples, be from about20 to about 60 amino acids in length. In other examples, stalk extensionregions can be about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,greater amino acids in length, or any integer within or outside of thatrange. In some cases, each stalk extension region has a sequence whichhas at least about 60% identity to the stalk region. In some examples,each stalk extension region has a sequence which has at least about 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater identity tothe stalk region.

In one embodiment, the stalk region is proximal to the membrane regionas depicted in FIG. 1A. In another embodiment, the stalk region isdistal to the membrane region as depicted in FIG. 1B.

In one embodiment, the stalk region and stalk extension region(s) can bederived or designed from a polypeptide of natural or of syntheticorigin. The stalk region and/or stalk extension region(s) can comprisehinge domain(s) derived from a cell surface protein or derivatives orvariants thereof. In some embodiments, the stalk region and/or stalkextension region(s) can comprise a hinge domain derived from CD28 orCD8alpha (CD8α). In some embodiments, each of the stalk region and stalkextension region(s) can be derived from at least one of a CD8alpha hingedomain, a CD28 hinge domain, a CTLA-4 hinge domain, a LNGFRextracellular domain, IgG1 hinge, IgG4 hinge and CH2-CH3 domain. Thestalk and stalk extension region(s) can be separately derived from anycombination of CD8alpha hinge domain, CD28 hinge domain, CTLA-4 hingedomain, LNGFR extracellular domain, IgG1 hinge, IgG4 hinge or CH2-CH3domain. As an example, the stalk region can be derived from CD8alphahinge domain and at least one stalk extension region can be derived fromCD28 hinge domain thus creating a hybrid spacer. As another example, thestalk region can be derived from an IgG1 hinge or IgG4 hinge and atleast one stalk extension region can be derived from a CH2-CH3 domain ofIgG.

In certain embodiments, the stalk region may comprise one or moredimerization sites to form homo or hetero dimerized chimericpolypeptides. In other embodiments, the stalk region or one or morestalk extension regions may contain mutations that eliminatedimerization sites altogether. In some embodiments, a stalk extensionregion(s) can contain at least one fewer dimerization site as comparedto a stalk region. For example, if a stalk region comprises twodimerization sites, a stalk extension region can comprise one or zerodimerization sites. As another example, if a stalk region comprises onedimerization site, a stalk extension region can comprise zerodimerization sites. In some examples, the stalk extension region(s)lacks a dimerization site.

Polypeptides

Disclosed herein are polypeptides that can be used with spacersdescribed herein. In one embodiment, such polypeptides that comprise spacers described herein are polypeptides that do not express on the cellmembrane surface or polypeptides that are unable to bind their targetdue to lack of proximity or steric hindrance. Examples of polypeptidesinclude, but are not limited to, ligands, ligand binding receptors,peptides, antibodies or antigen binding fragments thereof, such as Fab,Fab′, F(ab)2, and Fv fragments, fragments comprised of one or more CDRs,single-chain antibodies (e.g., single chain Fv fragments (scFv)),disulfide stabilized (dsFv) Fv fragments, heteroconjugate antibodies(e.g., bispecific antibodies), pFv fragments, heavy chain monomers ordimers, light chain monomers or dimers, and dimers consisting of oneheavy chain and one light chain, a chimeric antigen receptor (CAR).Antigen binding regions can also include ligand regions, for example aproliferation-inducing ligand (APRIL), a B cell-activating factor(BAFF), transmembrane activator and calcium-modulator and cyclophilinligand interactor (TACI), or a synthetically derived peptide.

In some embodiments, the polypeptide is a chimeric polypeptide. Incertain instances, a chimeric polypeptide described herein is an antigenbinding polypeptide. In some embodiments the polypeptide is a chimericantigen receptor (CAR). A polypeptide such as a CAR, as describedherein, comprises an antigen-binding region, a transmembrane region, anda spacer connecting said trans-membrane region with said antigen bindingregion. In one embodiment, the spacer comprises a stalk regioncomprising at least one dimerization site, and a stalk extension region.In other embodiments, said stalk extension region can comprise fewerdimerization sites as compared to said stalk region. In certain cases, achimeric polypeptide described herein, also comprises an intracellularsignaling domain. In some cases, the chimeric polypeptide does notcomprise an intracellular signaling domain. In certain cases, anintracellular signaling domain is expressed on as a separate polypeptidein an engineered cell expressing a chimeric polypeptide describedherein.

Additionally disclosed herein are antigen-binding polypeptidescomprising an antigen-binding region, a transmembrane region, and aspacer region connecting said trans-membrane region with said antigenbinding region, wherein said spacer region can comprise a stalk region(designated as “s”) and stalk extension region(s), which is hereindesignated as “s-n” as discussed herein.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and one stalk extension region, i.e.s′-n, wherein n=1. In some cases, the stalk extension region has asequence which has at least 60% identity to the stalk region. In otherexamples, the stalk extension regions has a sequence which has at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the stalkregion.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and two stalk extension region, i.e.s′-n, wherein n=2. In some cases, each of the stalk extension regionshas a sequence which has at least 60% identity to the stalk region. Inother examples, each of the stalk extension regions has a sequence whichhas at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identityto the stalk region.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and three stalk extension region,i.e. s′-n, wherein n=3. In some cases, each of the stalk extensionregions has a sequence which has at least 60% identity to the stalkregion. In other examples, each of the stalk extension regions has asequence which has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the stalk region.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and four stalk extension region,i.e. s′-n, wherein n=4. In some cases, each of the stalk extensionregions has a sequence which has at least 60% identity to the stalkregion. In other examples, each of the stalk extension regions has asequence which has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the stalk region.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and five stalk extension region,i.e. s′-n, wherein n=5. In some cases, each of the stalk extensionregions has a sequence which has at least 60% identity to the stalkregion. In other examples, each of the stalk extension regions has asequence which has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the stalk region.

Antigen binding polypeptides comprising a spacer are disclosed, whereinthe spacer comprises a stalk region and a stalk extension region,wherein the stalk extension region comprises more than 5 stalk extensionregions, i.e. s′-n, wherein n>5. In such cases, the stalk extensionregion can comprise n=6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, or more stalk extension regions. In some cases, each of saidstalk extension regions has a sequence which has at least 60% identityto the stalk region. In other examples, each of said stalk extensionregions has a sequence which has at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or greater identity to the stalk region. In some cases, thespacer comprises a peptide sequence with at least about 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the CD8α sequence shown in SEQ IDNO: 1. In some cases, the spacer comprises a peptide sequence encoded bya nucleotide sequence with at least about 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 25%, 26%, 27%,28%, 29%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the CD8α nucleotide sequence shown in SEQ ID NO:107.

In some aspects of the embodiments disclosed herein, a stalk region of asubject antigen binding polypeptide comprises a sequence with at leastabout 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to aCD8alpha hinge domain. A CD8α hinge domain can comprise a peptidesequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greateridentity to the sequence shown in SEQ ID NO: 3 or a peptide sequenceencoded by a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the sequence shown in SEQ ID NO:109. In some cases, a stalk extension region comprises a peptidesequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greateridentity to the sequence shown in SEQ ID NO: 2 or a peptide sequenceencoded by a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%,90%, 95%, or greater identity to the sequence shown in SEQ ID NO: 108.In some examples, a stalk region and stalk extension region can togethercomprise a peptide sequence with at least 65%, 70%, 75%, 80%, 85%, 90%,95% or greater identity to the sequence shown in SEQ ID NO: 4, 5, 6 or7, or a peptide sequence encoded by a nucleotide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to the sequenceshown in SEQ ID NO: 110, 111, 112 or 113.

In some embodiment, the stalk region and the stalk extension region(s)can be connected via a linker, such as whitlow linker (SEQ ID NO: 8 andSEQ ID NO: 114), GSG linker (SEQ ID NO: 9 and SEQ ID NO: 115), SGSGlinker (SEQ ID NO: 10) or (G4S)3 linker (SEQ ID NO: 11 and SEQ ID NO:117). In one embodiment, the CD8α hinge domain connected to (G4S)3linker (SEQ ID NO: 11) can comprise a peptide sequence shown in SEQ IDNO: 12 or a peptide sequence encoded by a nucleotide sequence shown inSEQ ID NO: 118. In another embodiment, the CD8α hinge domain connectedto whitlow linker can comprise a peptide sequence shown in SEQ ID NO: 13or a peptide sequence encoded by a nucleotide sequence shown in SEQ IDNO: 119. In yet another embodiment, the CD8α hinge domain connected towhitlow linker (2×) can comprise a peptide sequence shown in SEQ ID NO:14 or a peptide sequence encoded by a nucleotide sequence shown in SEQID NO: 120. In another embodiment, the CD8α hinge domain connected towhitlow linker (2×) can comprise a peptide sequence shown in SEQ ID NO:15 or a peptide sequence encoded by a nucleotide sequence shown in SEQID NO: 121.

In some cases, the spacer comprises a peptide sequence with at leastabout 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the CD28sequence shown in SEQ ID NO: 31. In some cases, the spacer comprises apeptide sequence encoded by a nucleotide sequence with at least about10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or greater identity to the CD28 nucleotidesequence shown in SEQ ID NO: 137. In some aspects of at least oneembodiment disclosed herein, a stalk region of a polypeptide describedherein comprises a sequence with at least about 65%, 70%, 75%, 80%, 85%,90%, 95%, or greater identity to a CD28 hinge domain. A CD28 hingedomain can comprise a peptide sequence with at least 65%, 70%, 75%, 80%,85%, 90%, 95%, or greater identity to the sequence shown in SEQ ID NO:32 or a peptide sequence encoded by a nucleotide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to thesequence shown in SEQ ID NO: 138. In some examples, a stalk region andstalk extension region can together comprise a peptide sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to thesequence shown in SEQ ID NO: 33, 34 or 35, or a peptide sequence encodedby a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or greater identity to the sequence shown in SEQ ID NO: 139,SEQ ID NO: 140 or SEQ ID NO: 141.

In some aspects of the embodiments disclosed herein, a stalk region of apolypeptide described herein comprises a sequence with at least about65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to a CTLA-4extracellular domain. In some aspects, the CTLA-4 extracellular domainmay comprise a partial sequence. In some cases, the CTLA-4 partialsequence comprises a peptide sequence with at least about 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the CTLA-4 sequence shown in SEQ IDNO: 36. In some cases, the spacer comprises a peptide sequence encodedby a nucleotide sequence with at least about 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 25%, 26%,27%, 28%, 29%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99% or greater identity to the CTLA-4 nucleotide sequence shown in SEQID NO: 142. A CTLA-4 extracellular domain can comprise a sequence withat least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to thesequence shown in SEQ ID NO: 37. In some cases, a stalk extension regioncomprises a sequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater identity to the sequence shown in SEQ ID NO: 37. In someexamples, a stalk region and stalk extension region can togethercomprise a sequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater identity to the sequence shown in SEQ ID NO: 38, SEQ ID NO: 39or SEQ ID NO: 40.

In some aspects of the embodiments disclosed herein, a stalk region or astalk extension region of a polypeptide described herein comprises asequence with at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, orgreater identity to a full length LNGFR extracellular domain (ECD). Insome cases, the LNGFR ECD is incapable of dimerization. A LNGFR ECD cancomprise a peptide sequence with at least 65%, 70%, 75%, 80%, 85%, 90%,95%, or greater identity to the sequence shown in SEQ ID NO: 16 or apeptide sequence encoded by a nucleotide sequence with at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the sequenceshown in SEQ ID NO: 122. In some cases, a stalk extension regioncomprises a peptide sequence with at least 65%, 70%, 75%, 80%, 85%, 90%,95%, or greater identity to the sequence shown in SEQ ID NO: 16 or apeptide sequence encoded by a nucleotide sequence with at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the sequenceshown in SEQ ID NO: 122. In some examples, a stalk region and stalkextension region can together comprise a peptide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to thesequence shown in SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ IDNO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ ID NO: 23, or a peptidesequence encoded by a nucleotide sequence with at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or greater identity to the sequence shown in SEQID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO:127, SEQ ID NO: 128 or SEQ ID NO: 129.

In some cases, a stalk extension region comprises a sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to thesequence shown in SEQ ID NO: 72 or SEQ ID NO: 73 or a peptide sequenceencoded by a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the sequence shown in SEQ ID NO:166 or SEQ ID NO: 167. In some examples, a stalk region and stalkextension region can together comprise a peptide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to the sequenceshown in SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79, or a peptidesequence encoded by a nucleotide sequence with at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or greater identity to the sequence shown in SEQID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173. In some examples, a stalkregion and stalk extension region can together comprise a peptidesequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or greateridentity to the sequence shown in SEQ ID NO: 80, SEQ ID NO: 81 or SEQ IDNO: 82, or a peptide sequence encoded by a nucleotide sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to thesequence shown in SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 176.

In some embodiment, the stalk region and the stalk extension region(s)can be connected via a linker, such as whitlow linker (SEQ ID NO: 8 andSEQ ID NO: 114), GSG linker (SEQ ID NO: 9 and SEQ ID NO: 115), SGSGlinker (SEQ ID NO: 10) or (G4S)3 linker (SEQ ID NO: 11 and SEQ ID NO:117). In one embodiment, the TCRa hinge domain connected to (G4S)3linker (SEQ ID NO: 11) can comprise a peptide sequence shown in SEQ IDNO: 74 or a peptide sequence encoded by a nucleotide sequence shown inSEQ ID NO: 168. In one embodiment, the TCRP hinge domain connected to(G4S)3 linker (SEQ ID NO: 11) can comprise a peptide sequence shown inSEQ ID NO: 75 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 169. In yet another embodiment, the TCRP hingedomain connected to whitlow linker can comprise a peptide sequence shownin SEQ ID NO: 76 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 170.

In some cases, a stalk extension region comprises a sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to thesequence shown in SEQ ID NO: 86 or SEQ ID NO: 87 or a peptide sequenceencoded by a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the sequence shown in SEQ ID NO:180 or SEQ ID NO: 181. In some examples, a stalk region and stalkextension region can together comprise a peptide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to the sequenceshown in SEQ ID NO: 91, SEQ ID NO: 92 or SEQ ID NO: 93, or a peptidesequence encoded by a nucleotide sequence with at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or greater identity to the sequence shown in SEQID NO: 185, SEQ ID NO: 186 or SEQ ID NO: 187. In some examples, a stalkregion and stalk extension region can together comprise a peptidesequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or greateridentity to the sequence shown in SEQ ID NO: 94, SEQ ID NO: 95 or SEQ IDNO: 96, or a peptide sequence encoded by a nucleotide sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to thesequence shown in SEQ ID NO: 188, SEQ ID NO: 189 or SEQ ID NO: 190.

In some embodiment, the stalk region and the stalk extension region(s)can be connected via a linker, such as whitlow linker (SEQ ID NO: 8 andSEQ ID NO: 114), GSG linker (SEQ ID NO: 9 and SEQ ID NO: 115), SGSGlinker (SEQ ID NO: 10) or (G4S)3 linker (SEQ ID NO: 11 and SEQ ID NO:117). In one embodiment, the TCRβ hinge domain connected to (G4S)3linker (SEQ ID NO: 11) can comprise a peptide sequence shown in SEQ IDNO: 88 or a peptide sequence encoded by a nucleotide sequence shown inSEQ ID NO: 182. In one embodiment, the TCRβ hinge domain connected to(G4S)3 linker (SEQ ID NO: 11) can comprise a peptide sequence shown inSEQ ID NO: 89 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 183. In yet another embodiment, the TCRβ hingedomain connected to whitlow linker can comprise a peptide sequence shownin SEQ ID NO: 90 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 184.

In some aspects of the embodiments disclosed herein, a dimerization sitecomprises a cysteine. In some cases, a dimerization site in a stalkregion described herein comprises two cysteines. In some aspects, thedimerization site can be ligand induced.

Additionally disclosed herein are polynucleotides encoding any of thepolypeptides disclosed herein, as well as vectors comprising one or moreof said polynucleotides. Vectors can be cloning vectors, deliveryvectors, expression vectors, or any combination thereof. Such vectorscan be viral vectors or non-viral vectors. For example, a vector can bea lentivirus vector, retroviral vector, adenoviral vector,adeno-associated viral vector, a Sleeping Beauty transposon, AttSite™Recombinase, PiggyBac™ transposon or other non-viral vector.

In some cases, an antigen-binding polypeptide comprising spacers withstalk extension region(s) as disclosed herein can have increasedantigen-binding compared to an otherwise identical antigen-bindingpolypeptide which lacks the stalk extension region(s). Antigen-bindingof the antigen-binding polypeptide comprising the stalk extensionregion(s) can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%500%, 1000% or greater as compared to an otherwise identicalantigen-binding polypeptide which lacks the stalk extension region.

Antigen-binding can be assessed by flow cytometry or a cell based assayor any other equivalent assay. Cell based assays may utilize a cell typeexpressing antigen of interest on the surface to assess antigen-binding.An antigen or a fragment thereof expressed as a soluble protein can beutilized to assess antigen-binding using flow cytometry or similarassay. Improvements in antigen-binding may be indirectly assessed byfunctional measurement of antigen-binding polypeptide or a chimericreceptor. For example, improved antigen-binding of a chimeric receptoror a CAR, as described herein, can be measured by increased specificcytotoxicity against target cells expressing the antigen.

In some cases, a polypeptide as disclosed herein can have increasedexpression on a cell surface compared to an otherwise identicalpolypeptide which lacks one or more stalk extension region(s). Cellsurface expression of the polypeptide comprising the stalk extensionregion(s) can be increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%500%, 1000% or greater as compared to an otherwise identical polypeptidewhich lacks the stalk extension region.

Cell surface expression level of a polypeptide of the present disclosurecan be assessed, for example, using a flow cytometry based assay.Improved expression of an antigen-binding polypeptide can be measured aspercentage of analyzed cells expressing said antigen-binding polypeptideor alternatively as average density of said antigen-binding polypeptideon the surface of a cell. Additional suitable methods that can be usedfor assessing cell surface expression of the antigen-bindingpolypeptides described herein include western blotting or any otherequivalent assay.

Chimeric Receptors

Polypeptides disclosed herein can be expressed in a modified effectorcell. In some embodiments, a modified effector cell comprises a chimericreceptor expressed on the surface of the cell. In some instances, thechimeric receptor comprises an antigen binding region that enablesrecognition and binding to a tumor antigen, e.g., a tumor-associatedantigen or a tumor-specific antigen. In some instances, the antigenbinding region comprises an antibody or binding fragment, for example,an Fab, an Fab′, an F(ab′)₂, an F(ab′)₃, an scFv, an sc(Fv)₂, a dsFv, adiabody, a minibody, and a nanobody or binding fragments thereof. Insome cases, the antigen binding region comprises an scFv. In some cases,the antigen-binding polypeptide is a chimeric antigen receptor (CAR)that comprises a scFv as an antigen binding domain. In some instances,the chimeric antigen receptor comprises a pattern-recognition receptor.In other cases, the chimeric receptor comprises an engineered T-cellreceptor (TCR).

Chimeric Antigen Receptors (CARs)

Polypeptides disclosed herein can comprise a chimeric antigen receptor(CAR). A CAR is an engineered receptor which grafts an exogenousspecificity onto an immune effector cell.

In some cases, a CAR disclosed herein comprises a spacer regionconnecting a transmembrane region with an antigen binding region. Insome cases, a spacer region of a CAR disclosed herein comprises 1) astalk region and 2) stalk extension region(s) adjacent to said stalkregion. Illustrative embodiments are described in FIGS. 1 and 2 . Insome embodiments, a CAR disclosed herein incorporates a spacer thatcomprises a stalk region and a stalk extension region (s′-n, whereinn=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20).

In some instances, a CAR comprises an extracellular region (ectodomain)that comprises an antigen binding region, a stalk region, a stalkextension region, a transmembrane region and, optionally anintracellular (endodomain) region. In some instances, the intracellularregion further comprises one or more intracellular signaling regions ordomains. In some instances, a CAR described herein comprises an antigenbinding region, a stalk region, a stalk extension region, atransmembrane region, one or more costimulatory regions or domains, anda signaling region for T-cell activation.

An antigen binding region can comprise complementary determining regionsof a monoclonal antibody, variable regions of a monoclonal antibody,and/or antigen binding fragments thereof. A complementarity determiningregion (CDR) is a short amino acid sequence found in the variabledomains of antigen receptor (e.g., immunoglobulin and T-cell receptor)proteins that complements an antigen and therefore provides the receptorwith its specificity for that particular antigen. Each polypeptide chainof an antigen receptor can contain three CDRs (CDR1, CDR2, and CDR3). Insome instances, an antigen binding region comprises F(ab′)2, Fab′, Fab,Fv, or scFv. In some cases, an antigen binding region is an scFv. Insome cases, an antigen binding region is a Fab. In some cases, anantigen binding region is a Fab′. In some cases, an antigen bindingregion is F(ab′)2. In some cases, an antigen binding region is an Fv.

In some embodiments, a CAR or a chimeric receptor or antigen bindingpolypeptide described herein comprises an antigen binding region thatbinds to an epitope on CD19, BCMA, CD44, α-Folate receptor, CAIX, CD30,ROR1, CEA, EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3,IL-13Ra2, KDR, EDB-F, mesothelin, GPC3, CSPG4, HER1/HER3, HER2, CD44v6,CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP, c-MET, PSCA, CS1, CD38,IL-11Rα, EphA2, CLL-1, CD22, EGFR, Folate receptor α, Mucins such asMUC-1 or MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFR, CD20, EGFRvIII,CD123 or VEGF-R2. In some embodiments, a CAR or a chimeric receptor orantigen binding polypeptide described herein comprises an antigenbinding region that binds to an epitope on CD19, CD33, BCMA, CD44,α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3,Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin,GPC3, CSPG4, HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20, CD174, CD138,L1-CAM, FAP, c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2, CLL-1, CD22, EGFR,Mucins such as MUC-1 or MUC-16, MAGE-A1, h5T4, PSMA, TAG-72, EGFRvIII,CD123 and VEGF-R2. In some embodiments, a CAR or a chimeric receptor orantigen binding polypeptide described herein comprises an antigenbinding region that binds to an epitope on CD19 or CD33. In someinstances, a CAR or a chimeric receptor or antigen binding polypeptidedescribed herein comprises an antigen binding region that binds to anepitope on CD19. In some cases, a CAR or a chimeric receptor or antigenbinding polypeptide described herein comprises an antigen binding regionthat binds to an epitope on CD33. In some cases, a CAR or a chimericreceptor or antigen binding polypeptide described herein comprises anantigen binding region that binds to an epitope on ROR-1. In some cases,a CAR or a chimeric receptor or antigen binding polypeptide describedherein comprises an antigen binding region that binds to an epitope onEGFRvIII. In further embodiments, a CAR or a chimeric receptor orantigen binding polypeptide described herein comprises an autoantigen oran antigen binding region that binds to an epitope on HLA-A2, myelinoligodendrocyte glycoprotein (MOG), factor VIII (FVIII), MAdCAM1, SDF1,or collagen type II

In some embodiments, the polynucleotides, polypeptides and methodsdescribed herein can be used for the treatment of a hyperproliferativedisease, such as a cancer, an autoimmune disease or for the treatment ofan infection, such as a viral, bacterial or parasitic infection. In someaspects, the antigen is an antigen that is elevated in cancer cells, inautoimmune cells or in cells that are infected by a virus, bacteria orparasite. Pathogens that may be targeted include, without limitation,Plasmodium, trypanosome, Aspergillus, Candida, Hepatitis A, Hepatitis B,Hepatitis C, HSV, HPV, RSV, EBV, CMV, JC virus, BK virus, or Ebolapathogens. Autoimmune diseases can include graft-versus-host disease,rheumatoid arthritis, lupus, celiac disease, Crohn's disease, SjogrenSyndrome, polymyalgia rheumatic, multiple sclerosis, neuromyelitisoptica, ankylosing spondylitis, Type 1 diabetes, alopecia areata,vasculitis, temporal arteritis, bullous pemphigoid, psoriasis, pemphigusvulgaris or autoimmune uveitis.

The pathogen recognized by a CAR may be essentially any kind ofpathogen, but in some embodiments the pathogen is a fungus, bacteria, orvirus. Exemplary viral pathogens include those of the families ofAdenoviridae, Epstein-Barr virus (EBV), Cytomegalovirus (CMV),Respiratory Syncytial Virus (RSV), JC virus, BK virus, HPV, HSV, HHVfamily of viruses, Hepatitis family of viruses, Picornaviridae,Herpesviridae, Hepadnaviridae, Flaviviridae, Retroviridae,Orthomyxoviridae, Paramyxoviridae, Papovaviridae, Polyomavirus,Rhabdoviridae, and Togaviridae. Exemplary pathogenic viruses causesmallpox, influenza, mumps, measles, chickenpox, ebola, and rubella.Exemplary pathogenic fungi include Candida, Aspergillus, Cryptococcus,Histoplasma, Pneumocystis, and Stachybotrys. Exemplary pathogenicbacteria include Streptococcus, Pseudomonas, Shigella, Campylobacter,Staphylococcus, Helicobacter, E. coli, Rickettsia, Bacillus, Bordetella,Chlamydia, Spirochetes, and Salmonella. In some embodiments the pathogenreceptor Dectin-1 may be used to generate a CAR that recognizes thecarbohydrate structure on the cell wall of fungi such as Aspergillus. Inanother embodiment, CARs can be made based on an antibody recognizingviral determinants (e.g., the glycoproteins from CMV and Ebola) tointerrupt viral infections and pathology.

In some embodiments, a spacer region as described herein can be used tolink the antigen-binding region to the transmembrane region of a CAR. Insome instances, a spacer can comprise any oligonucleotide- orpolypeptide that functions to link the transmembrane region to, eitherthe extracellular region or, the cytoplasmic region in the polypeptidechain. In some embodiments, the spacer is flexible enough to allow theantigen-binding region to orient in different directions to facilitateantigen recognition.

As described herein, a spacer region can comprise a stalk region andstalk extension region(s). In some instances, the stalk region comprisesthe hinge region from IgG1, or the stalk region comprises a sequencewith at least 80% homology to the hinge region from IgG1. In alternativeinstances, the stalk region comprises IgG3 hinge region or a sequencewith at least 80% homology to the IgG3 hinge region (SEQ ID NO: 41). Inalternative instances, the stalk region comprises IgG4 hinge region or asequence with at least 80% homology to the IgG4 hinge region (SEQ ID NO:42). In other cases, a stalk region comprises a peptide sequence with atleast about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater homologyto a peptide sequence shown in SEQ ID NO: 43, 44, 45, 46, 47 or 48. Inanother case, a stalk region comprises a peptide sequence encoded by anucleotide sequence with at least about 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or greater identity to the sequence shown in SEQ ID NO: 144. Insome cases, the stalk region comprises a CD8α hinge region, or asequence with at least 80% homology to the hinge region of CD8α. Forexample, the stalk region can comprise a sequence with at least 80%,85%, 90%, 95%, or greater than 95% homology to the hinge region of CD8α.In some cases, the stalk region comprises a CD28 hinge region, or asequence with at least 80% homology to the hinge region of CD28. Forexample, the stalk region can comprise a sequence with at least 80%,85%, 90%, 95%, or greater than 95% homology to the hinge region of CD28.In some cases, the stalk region comprises an IgG4 12 amino acid hingeregion (ESKYGPPCPPCP (SEQ ID NO: 43)) or IgG4 hinge regions (SEQ ID NO:42) as described in WO/2016/073755.

In some embodiments, the stalk region comprises a dimerization site. Adimerization site can comprise a disulfide bond formation site. Adimerization site can comprise cysteine residue(s). A stalk region canbe capable of forming a disulfide bond. Such a disulfide bond can beformed at a disulfide bond forming site or a dimerization site. In someexamples, the dimerization occurs between the stalk region of a firstCAR and a homologous stalk region of a homologous second CAR.

In some embodiments, a stalk extension region is used to link theantigen-binding region to the stalk region. In additional embodiments, astalk extension region is used to link the stalk region to thetransmembrane region of the CAR. For instance, when the stalk regioncomprises a hinge domain derived from an IgG, a non-Fc CH2 or CH3 domaincan be used as a stalk extension region. In another embodiment, thestalk region and the stalk extension region(s) can be connected via alinker. In other embodiments, one stalk extension region can beconnected to another stalk extension region via a linker. Examples ofsuch linkers can include a glycine-serine rich linker. In oneembodiment, a stalk region or a stalk extension region of a polypeptidedescribed herein comprises a peptide sequence with at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the IgG4hinge-CH2-CH3 spacer sequence shown in SEQ ID NO: 49. In some cases, astalk region or a stalk extension region of a polypeptide describedherein comprises a peptide sequence encoded by a nucleotide sequencewith at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the IgG4 hinge-CH2-CH3 spacer nucleotide sequenceshown in SEQ ID NO: 145. In one embodiment, a stalk region or a stalkextension region of a polypeptide described herein comprises a peptidesequence with at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or greater identity to the IgG4 hinge-CH3 spacer sequence shown in SEQID NO: 50.

In some instances, the stalk extension domain comprises a sequence thatis partially homologous to the stalk region. In some instances, each ofthe stalk extension region comprises a sequence that is homologous tothe stalk region, except that the stalk extension region lacks thedimerization site of the stalk region. In some cases, each of the stalkextension regions comprises a sequence identical to the stalk region. Inother cases, each of the stalk extension regions comprises a sequenceidentical to the stalk region with at least one amino acid residuesubstitution relative to the stalk region. In some cases, each of thestalk extension region is not capable of forming a disulfide bond or isnot capable of dimerization with a homologous stalk extension region.

In embodiments described herein, a polypeptide can comprise atransmembrane region or transmembrane domain that can be derived fromeither a natural or a synthetic source. Where the source is natural, theregion can be derived from any membrane-bound or transmembrane protein.Suitable transmembrane regions can include, but not limited to, thetransmembrane region(s) of alpha, beta or zeta chain of the T-cellreceptor; or a transmembrane region from CD28, CD3 epsilon, CD3ζ, CD45,CD4, CD5, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86,CD134, CD137, CD152 (CTLA-4) or CD154. Alternatively, the transmembraneregion or domain can be synthetic, and can comprise hydrophobic residuessuch as leucine and valine. In some embodiments, a triplet ofphenylalanine, tryptophan and valine is found at one or both termini ofa synthetic transmembrane domain. Optionally, a short oligonucleotide orpolypeptide linker, in some embodiments, between 2 and 10 amino acids inlength may form the linkage between the transmembrane domain and thecytoplasmic signaling domain of a CAR. In some embodiments, the linkeris a glycine-serine linker.

In some embodiments, the transmembrane region comprises a CD8αtransmembrane domain, a CD152 (CTAL-4), TCRγ1, TCRδ or a CD3ζtransmembrane domain. In some embodiments, the transmembrane regioncomprises a CD8α transmembrane domain (SEQ ID NO: 24 and SEQ ID NO:130). In other embodiments, the transmembrane region comprises a CD3ζtransmembrane domain. In another embodiment, the transmembrane regioncomprises a CD152 (CTLA-4) transmembrane domain (SEQ ID NO: 25 and SEQID NO: 131). In yet another embodiment, the transmembrane regioncomprises a TCRα transmembrane domain (SEQ ID NO: 71 and SEQ ID NO:165), a TCRβ transmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), aTCRγ1 transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195). In someembodiments, the transmembrane region comprises a TCRδ transmembranedomain (SEQ ID NO: 106 and SEQ ID NO: 200).

The intracellular region or intracellular domain can comprise one ormore costimulatory domains. Exemplary costimulatory domains include, butare not limited to, CD3ζ, CD8, CD27, CD28, 4-1BB (CD137), ICOS, DAP10,DAP12, OX40 (CD134) or fragment or combination thereof. In someinstances, a CAR described herein comprises one or more, or two or moreof costimulatory domains selected from CD3ζ, CD8, CD27, CD28, 4-1BB(CD137), ICOS, DAP10, DAP 12, OX40 (CD134) or fragment or combinationthereof. In some instances, a CAR described herein comprises one ormore, or two or more of costimulatory domains selected from CD3ζ, CD27,CD28, 4-1BB (CD137), ICOS, OX40 (CD134) or fragment or combinationthereof. In some instances, a CAR described herein comprises one ormore, or two or more of costimulatory domains selected from CD3ζ, CD8,CD28, 4-1BB (CD137), or fragment or combination thereof. In someinstances, a CAR described herein comprises one or more, or two or moreof costimulatory domains selected from CD3ζ, CD28, 4-1BB (CD137), orfragment or combination thereof. In some instances, a CAR describedherein comprises costimulatory domains CD3ζ, CD28 and 4-1BB (CD137) ortheir respective fragments thereof. In some instances, a CAR describedherein comprises costimulatory domains CD28 and OX40 (CD134) or theirrespective fragments thereof. In some instances, a CAR described hereincomprises costimulatory domains CD8 and CD28 or their respectivefragments thereof. In some instances, a CAR described herein comprisescostimulatory domain CD28 (SEQ ID NO: 26 and SEQ ID NO: 132) or afragment thereof. In some instances, a CAR described herein comprisescostimulatory domain 4-1BB (CD137) (SEQ ID NO: 27 and SEQ ID NO: 133) ora fragment thereof. In some instances, a CAR described herein comprisescostimulatory domain OX40 (CD134) or a fragment thereof. In someinstances, a CAR described herein comprises costimulatory domain CD8 ora fragment thereof. In some instances, a CAR described herein comprisescostimulatory domain CD3 (SEQ ID NO: 28 and SEQ ID NO: 134) or afragment thereof.

In some embodiments, the intracellular region or intracellular domainfurther comprises a signaling domain for T-cell activation. In someinstances, the signaling domain for T-cell activation comprises a domainderived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CD5, CD22, CD79a, CD79b or CD66d. In some cases, the signalingdomain for T-cell activation comprises a domain derived from CD3ζ.

CD19-Specific CARs

CD19 is a cell surface glycoprotein of the immunoglobulin superfamilyand is found predominately in malignant B-lineage cells. In someinstances, CD19 has also been detected in solid tumors such aspancreatic cancer, liver cancer, and prostate cancer.

In some embodiments, described herein include a CD19-specific CAR, inwhich the antigen binding region comprises a F(ab′)2, Fab′, Fab, Fv, orscFv. In some instances, the antigen binding region recognizes anepitope on CD19.

In some embodiments, an antigen binding region encompassed by apolypeptide described herein recognizes an epitope on CD19 that is alsorecognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (JunoTherapeutics). In some embodiments, CD19 comprises a peptide sequence atleast 80% homology to the CD19 sequence shown in SEQ ID NO: 51. In someembodiments, described herein include a CD19-specific CAR expressed onan effector cell such as a T cell, in which the antigen binding regionrecognizes an epitope on CD19 that is also recognized by JCAR014,JCAR015, JCAR017, or 19-28z CAR (Juno Therapeutics). In some instances,the CD19-specific CAR is encompassed by a polypeptide which furthercomprises a transmembrane region or transmembrane domain selected from aCD8alpha transmembrane domain (SEQ ID NO: 24 and SEQ ID NO: 130) or aCD3ζ transmembrane domain; one or more costimulatory domains selectedfrom CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) orfragment or combination thereof; and a signaling region or signalingdomain from CD3ζ. In some instances, the CD19-specific CAR is expressedas part of a polypeptide which further comprises a stalk region and astalk extension region as disclosed herein. For example, theCD19-specific CAR comprising polypeptide can further comprise a stalkregion comprising a CD8α hinge region, and a stalk extension region(s′-n), wherein n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, or 20, wherein each stalk extension region beinghomologous to a CD8α hinge region except for lacking a dimerizationsite.

In some embodiments, a CD19-specific CAR encompassed by a polypeptidedescribed herein comprises an scFv antigen binding region, and theantigen binding region recognizes an epitope on CD19 that is alsorecognized by JCAR014, JCAR015, JCAR017, or 19-28z CAR (JunoTherapeutics). In some instances, the CD19-specific CAR is encompassedby a polypeptide that further comprises a transmembrane domain selectedfrom a CD8alpha transmembrane domain (SEQ ID NO: 24 and SEQ ID NO: 130)or a CD3ζ transmembrane domain; one or more costimulatory domainsselected from CD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP 12, OX40(CD134) or fragment or combination thereof; and a signaling domain fromCD3ζ. In some cases, the CD19-specific CAR is encompassed by apolypeptide that further comprises a transmembrane domain selected froma CD152 (CTLA-4) transmembrane domain (SEQ ID NO: 25 and SEQ ID NO:131), a TCRα transmembrane domain (SEQ ID NO: 71 and SEQ ID NO: 165), aTCRβ transmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), a TCRγ1transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195) or a TCRδtransmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200). In someinstances, the polypeptide comprising a CD19-specific CAR cell furthercomprises a stalk region and a stalk extension region as disclosedherein. For example, the polypeptide can further comprise a stalk regioncomprising a CD8α hinge region, and a stalk extension region (s′-n),wherein n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, or 20, wherein each stalk extension region being homologous to aCD8α hinge region except for lacking a dimerization site.

In some embodiments, a CD19-specific CAR expressed on an effector cellsuch as a T cell described herein comprises an anti-CD19 antibodydescribed in US20160152723.

In some embodiments, an antigen binding region encompassed by apolypeptide described herein recognizes an epitope on CD19 that is alsorecognized by KTE-C19 (Kite Pharma, Inc.). In some embodiments,described herein include a CD19-specific CAR-T cell, in which theantigen binding region recognizes an epitope on CD19 that is alsorecognized by KTE-C19. In some instances, the CD19-specific CAR furthercomprises a transmembrane domain selected from a CD8alpha transmembranedomain (SEQ ID NO: 24 and SEQ ID NO: 130) or a CD3ζ transmembranedomain; one or more costimulatory domains selected from CD27, CD28,4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragment orcombination thereof; and a signaling domain from CD3ζ. In some cases,the CD19-specific CAR is encompassed by a polypeptide that furthercomprises a transmembrane domain selected from a CD152 (CTLA-4)transmembrane domain (SEQ ID NO: 25 and SEQ ID NO: 131), a TCRαtransmembrane domain (SEQ ID NO: 71 and SEQ ID NO: 165), a TCRβtransmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), a TCRγ1transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195) or a TCRδtransmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200).

Some embodiments, described herein include a CD19-specific CARcomprising an scFv antigen binding region, and the antigen bindingregion recognizes an epitope on CD19 that is also recognized by KTE-C19.In some instances, the CD19-specific CAR-T cell further comprises atransmembrane domain selected from a CD8alpha transmembrane domain (SEQID NO: 24 and SEQ ID NO: 130) or a CD3ζ transmembrane domain; one ormore costimulatory domains selected from CD27, CD28, 4-1BB (CD137),ICOS, DAP10, DAP12, OX40 (CD134) or fragment or combination thereof; anda signaling domain from CD3ζ.

In some embodiments, a CD19-specific CAR described herein comprises ananti-CD19 antibody described in WO2015187528 or fragment or derivativethereof.

In some embodiments, the antigen binding region recognizes an epitope onCD19 that is also recognized by CTL019 (Novartis). In some embodiments,the antigen binding region recognizes an epitope on CD19 that is alsorecognized by UCART19 (Cellectis). In some embodiments, the antigenbinding region recognizes an epitope on CD19 that is also recognized byBPX-401 (Bellicum). In some cases, the antigen binding region recognizesan epitope on CD19 that is also recognized by blinaturnormab (Angen),coltuximabravtansine (ImmunoGen IncISanofi-aventis), MOR208 (MorphosysAG/Xencor Inc.). MEDI-551 (Medimmune), denintuzumabmafodotin (SeattleGenetics), B4 (or DI-B34) (Merck Serono), taplitunmomaabpaptox (NationalCancer Institute), XmAb 5871 (Amgen/Xencor, Inc.). MDX-1342 (Medarex) orAFMI1 (Affimed). In some embodiments, described herein include aCD19-specific CAR expressed on an effector cell such as a T cell, inwhich the antigen binding region recognizes an epitope on CD19 that isalso recognized by CTL019. In some instances, the CD19-specific CARfurther comprises a transmembrane domain selected from a CD8alphatransmembrane domain (SEQ ID NO: 24 and SEQ ID NO: 130) or a CD3ζtransmembrane domain; one or more costimulatory domains selected fromCD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134) or fragmentor combination thereof; and a signaling domain from CD3ζ. In some cases,the CD19-specific CAR is encompassed by a polypeptide that furthercomprises a transmembrane domain selected from a CD152 (CTLA-4)transmembrane domain (SEQ ID NO: 25 and SEQ ID NO: 131), a TCRαtransmembrane domain (SEQ ID NO: 71 and SEQ ID NO: 165), a TCRβtransmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), a TCRγ1transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195) or a TCRδtransmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200). In someinstances, the CD19-specific CAR is encoded as part of a polypeptidethat further comprises a stalk region and a stalk extension region asdisclosed herein. For example, the CD19-specific CAR can be encompassedby a polypeptide that further comprise a stalk region comprising a CD8αhinge region, and a stalk extension region (s′-n), wherein n=0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, whereineach stalk extension region being homologous to a CD8α hinge regionexcept for lacking a dimerization site.

Some embodiments, described herein include a CD19-specific CAR expressedon an effector cell such as a T cell comprising an scFv antigen bindingregion, and the antigen binding region recognizes an epitope on CD19that is also recognized by at least one of CTL019, BPX-401, blinatumomab(Amgen), coltuximabravtansine (ImmunoGen Inc./Sanofi-aventis), MOR208(Morphosys AG/Xencor Inc.), MEDI-551 (Medimmine), denintuzumabmafodotin(Seattle Genetics). B4 (or DI-B4) (Merck Serono), taplitumomabpaptox(National Cancer Institute), XmAb 5871 (Amgen/Xencor, Inc.). MDX-1342(Medarex) and AFM11 (Affimed). In some instances, the CD19-specific CARis encompassed by a polypeptide which further comprises a transmembranedomain selected from a CD8alpha transmembrane domain (SEQ ID NO: 24 andSEQ ID NO: 130) or a CD3ζ transmembrane domain; one or morecostimulatory domains selected from CD27, CD28, 4-1BB (CD137), ICOS,DAP10, DAP12, OX40 (CD134) or fragment or combination thereof; and asignaling domain from CD3ζ. In some cases, the CD19-specific CAR isencompassed by a polypeptide that further comprises a transmembranedomain selected from a CD152 (CTLA-4) transmembrane domain (SEQ ID NO:25 and SEQ ID NO: 131), a TCRα transmembrane domain (SEQ ID NO: 71 andSEQ ID NO: 165), a TCRβ transmembrane domain (SEQ ID NO: 85 and SEQ IDNO: 179), a TCRγ1 transmembrane domain (SEQ ID NO: 101 and SEQ ID NO:195) or a TCRδ transmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200).In some cases, the CD19-specific CAR is encompassed by a polypeptidethat further comprises a signaling domain selected from a DAP10signaling domain (SEQ ID NO: 29 and SEQ ID NO: 135), or a DAP12signaling domain (SEQ ID NO: 30 and SEQ ID NO: 136). In some instances,a polypeptide comprising the CD19-specific CAR further comprises a stalkregion and a stalk extension region as disclosed herein. For example,the polypeptide can further comprise a stalk region comprising a CD8αhinge region, and a stalk extension region (s′-n), wherein n=0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, whereineach stalk extension region being homologous to a CD8α hinge regionexcept for lacking a dimerization site.

In some embodiments, the CD19-specific CAR described herein comprises ananti-CD19 monoclonal antibody variable light chain comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:53 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 147. In someembodiments, the CD19-specific CAR described herein comprises ananti-CD19 monoclonal antibody variable heavy chain comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:54 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 148. In someembodiments, the CD19-specific CAR described herein comprises ananti-CD19 scFv with Whitlow linker comprising a peptide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 55 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 149. In some embodiments, theCD19-specific CAR described herein comprises CD19 specific chimericantigen receptor with CD8-1× spacer (CD19-CD8α-CD28-CD3ζ) comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 56 or a peptide sequence encoded by a nucleotide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 150. In someembodiments, the CD19-specific CAR described herein comprises CD19specific chimeric antigen receptor with CD8-2× spacer comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 57 or a peptide sequence encoded by a nucleotide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 151. In someembodiments, the CD19-specific CAR described herein comprises CD19specific chimeric antigen receptor with CD8-3× spacer comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 58 or a peptide sequence encoded by a nucleotide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 152. In someembodiments, the CD19-specific CAR described herein comprises CD19specific chimeric antigen receptor with CD8-3× v2 spacer comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 59 or a peptide sequence encoded by a nucleotide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 153. In someembodiments, the CD19-specific CAR described herein comprises CD19specific chimeric antigen receptor with CD8-4× spacer comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 60 or a peptide sequence encoded by a nucleotide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 154.

CD33-Specific CARs

CD33/Siglec-3 is a restricted leukocyte antigen expressed specificallyin myeloid lineage cells. In some instances, CD33 has also been detectedin lymphoid cells.

In some embodiments, the disclosure herein includes a CD33-specific CAR,in which the antigen binding region comprises a F(ab′)₂. Fab′, Fab, Fv,or scFv that binds CD33.

In some embodiments, the antigen binding region recognizes an epitope onCD33 that is also recognized by Lintuzumab (Seattle Genetics), BI 836858(Boehringer Ingelheim). In some instances, a polypeptide describedherein comprises the CD33-specific CAR and further comprises atransmembrane region or transmembrane domain selected from a CD8alphatransmembrane domain (SEQ ID NO: 24 and SEQ ID NO: 130) or a CD3ζtransmembrane domain; one or more costimulatory domains selected fromCD27, CD28, 4-1BB (CD137), ICOS, DAP10, DAP12, OX40 (CD134), CD3-zeta orfragment or combination thereof; and a signaling region or signalingdomain from CD3ζ. In some cases, the CD33-specific CAR is encompassed bya polypeptide that further comprises a transmembrane domain selectedfrom a CD152 (CTLA-4) transmembrane domain (SEQ ID NO: 25 and SEQ ID NO:131), a TCRα transmembrane domain (SEQ ID NO: 71 and SEQ ID NO: 165), aTCRβ transmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), a TCRγ1transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195) or a TCRδtransmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200). In some cases,the CD33-specific CAR is encompassed by a polypeptide that furthercomprises a signaling domain selected from a DAP10 signaling domain (SEQID NO: 29 and SEQ ID NO: 135), or a DAP12 signaling domain (SEQ ID NO:30 and SEQ ID NO: 136). In some cases, the CD33-specific CAR isencompassed by a polypeptide that further comprises a signaling domainselected from a DAP10 signaling domain (SEQ ID NO: 29 and SEQ ID NO:135), or a DAP12 signaling domain (SEQ ID NO: 30 and SEQ ID NO: 136). Insome instances, the CD33-specific CAR further comprises a stalk regionand a stalk extension region as disclosed herein. For example, theCD33-specific CAR can further comprise a spacer wherein the spacercomprises a stalk region comprising a CD8α hinge region, and stalkextension region(s), s′-n, wherein n=0, 1, 2, 3 or 4.

In some embodiments, the CD33-specific CAR described herein comprises ananti-CD33 monoclonal antibody variable light chain comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:61 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 155. In someembodiments, the CD33-specific CAR described herein comprises ananti-CD33 monoclonal antibody variable heavy chain comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:62 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 156. In someembodiments, the CD33-specific CAR described herein comprises ananti-CD33 monoclonal antibody scFv comprising a peptide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 63 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 157. In some embodiments, theCD33-specific CAR described herein comprises CD33 specific chimericantigen receptor with CD8 1× spacer comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 64 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 158. In some embodiments,the CD33-specific CAR described herein comprises CD33 specific chimericantigen receptor with CD8 2× spacer comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 65 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 159. In some embodiments,the CD33-specific CAR described herein comprises CD33 specific chimericantigen receptor with CD8 3× spacer comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 66 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 160. In some embodiments,the CD33-specific CAR described herein comprises CD33 specific chimericantigen receptor with CD8 3× v2 spacer comprising a peptide sequencewith at least 80% homology to the sequence shown in SEQ ID NO: 67 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 161. In some embodiments,the CD33-specific CAR described herein comprises CD33 specific chimericantigen receptor with CD8 4× spacer comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 68 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 162.

EGFRvIII-Specific CARs

In another embodiment, a CAR described herein is a EGFRvIII specificCAR. “EGFRvIII”, “EGFR variant III”, “EGFR type III mutant”, “EGFR.D2-7”or “de2-7EGFR” is a mutated form of epidermal growth factor receptor(EGFR; ErbB-1; HER1), a transmembrane protein that is a receptor formembers of the epidermal growth factor (EGF) family of extracellularprotein ligands in human and non-human subjects. EGFRvIII ischaracterized by a deletion of exons 2-7 of the wild type EGFR gene,which results in an in-frame deletion of 267 amino acids in theextracellular domain of the full length wild type EGFR protein. EGFRvIIIalso contains a novel glycine residue inserted at the fusion junctioncompared to wild type EGFR. The truncated receptor EGFRvIII is unable tobind any known EGFR ligand; however, it shows constitutive tyrosinekinase activity. This constitutive activation is important to itspro-oncogenic effect. A kinase-deficient EGFRvIII is unable to confer asimilar oncogenic advantage. EGFRvIII is highly expressed inglioblastoma (GBM) and can be detected in some other solid tumor typesbut not in normal tissues.

In some embodiments, the antigen binding moiety of a CAR describedherein is specific to EGFRvIII (EGFRvIII CAR). The EGFRvIII-specificCAR, when expressed on the cell surface, redirects the specificity of Tcells to human EGFRvIII. In embodiments, the antigen binding domaincomprises a single chain antibody fragment (scFv) comprising a variabledomain light chain (VL) and variable domain heavy chain (VH) of a targetantigen specific monoclonal anti-EGFRvIII antibody joined by a flexiblelinker, such as a glycine-serine linker or a Whitlow linker. Inembodiments, the scFv is clone 139 (SEQ ID NO: 221 and 222). In someembodiments, the scFv is anti-EGFRvIII scFv clone MR1 (SEQ ID NO 223;SEQ ID NO 224), anti-EGFRvIII scFv clone MR1-1 (SEQ ID NO 225; SEQ ID NO226), anti-EGFRvIII scFv clone huMR1-1 (SEQ ID NO 227; SEQ ID NO 228),anti-EGFRvIII scFv clone huMR1-2 (SEQ ID NO 229; SEQ ID NO 230). In someembodiments, the antigen binding moiety may comprise VH and VL that aredirectionally linked, for example, from N to C terminus, VH-linker-VL orVL-linker-VH.

In embodiments, a CAR described herein comprises an antigen-bindingmoiety comprising a VL polypeptide having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acidsequence of SEQ ID NO 204 (anti-EGFRvIII clone 139 VL). In embodiments,a CAR described herein comprises an antigen-binding moiety comprising aVH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 202(anti-EGFRvIII clone 139 VH).

In embodiments, a CAR described herein comprises an antigen-bindingmoiety comprising a VL polypeptide having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acidsequence of SEQ ID NO 208 (anti-EGFRvIII clone MR1 VL). In embodiments,a CAR described herein comprises an antigen-binding moiety comprising aVH polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% identity with the amino acid sequence of SEQ ID NO 206(anti-EGFRvIII clone MR1 VH). In embodiments, a CAR described hereincomprises an antigen-binding moiety comprising a VL polypeptide havingat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity with the amino acid sequence of SEQ ID NO 212 (anti-EGFRvIIIclone MR1-1 VL). In embodiments, a CAR described herein comprises anantigen-binding moiety comprising a VH polypeptide having at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with theamino acid sequence of SEQ ID NO 210 (anti-EGFRvIII clone MR1-1 VH).

In embodiments, a CAR described herein comprises an antigen-bindingmoiety comprising a VL polypeptide having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acidsequence of SEQ ID NO 216 (anti-EGFRvIII clone humMR1-1 VL). Inembodiments, a CAR described herein comprises an antigen-binding moietycomprising a VH polypeptide having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence ofSEQ ID NO 214 (anti-EGFRvIII clone humMR1-1 VH). In embodiments, a CARdescribed herein comprises an antigen-binding moiety comprising a VLpolypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity with the amino acid sequence of SEQ ID NO 220(anti-EGFRvIII clone humMR1-2 VL). In embodiments, a CAR describedherein comprises an antigen-binding moiety comprising a VH polypeptidehaving at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%identity with the amino acid sequence of SEQ ID NO 218 (anti-EGFRvIIIclone humMR1-2 VH).

In some embodiments, the EGFRvIII-specific CAR described hereincomprises an anti-EGFRvIII monoclonal antibody scFv (clone 139)comprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 222 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:221. In some embodiments, the EGFRvIII-specific CAR described hereincomprises an anti-EGFRvIII monoclonal antibody scFv (MR1) comprising apeptide sequence with at least 80% homology to the sequence shown in SEQID NO: 224 or a peptide sequence encoded by a nucleotide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 223. In someembodiments, the EGFRvIII-specific CAR described herein comprises ananti-EGFRvIII monoclonal antibody scFv (MR1-1) comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:226 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 225. In someembodiments, the EGFRvIII-specific CAR described herein comprises ananti-EGFRvIII monoclonal antibody scFv (huMR1-1) comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:228 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 227. In someembodiments, the EGFRvIII-specific CAR described herein comprises ananti-EGFRvIII monoclonal antibody scFv (huMR1-2) comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:230 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 229.

In some instances, a polypeptide described herein comprises theEGFRvIII-specific CAR and further comprises a transmembrane region ortransmembrane domain selected from a CD8alpha transmembrane domain (SEQID NO: 24 and SEQ ID NO: 130) or a CD3ζ transmembrane domain; one ormore costimulatory domains selected from CD27, CD28, 4-1BB (CD137),ICOS, DAP10, DAP12, OX40 (CD134), CD3ζ or fragment or combinationthereof; and a signaling region or signaling domain from CD3ζ. In somecases, the EGFRvII-specific CAR is encompassed by a polypeptide thatfurther comprises a transmembrane domain selected from a CD152 (CTLA-4)transmembrane domain (SEQ ID NO: 25 and SEQ ID NO: 131), a TCRαtransmembrane domain (SEQ ID NO: 71 and SEQ ID NO: 165), a TCRβtransmembrane domain (SEQ ID NO: 85 and SEQ ID NO: 179), a TCRγ1transmembrane domain (SEQ ID NO: 101 and SEQ ID NO: 195) or a TCRδtransmembrane domain (SEQ ID NO: 106 and SEQ ID NO: 200). In some cases,the EGFRvIII-specific CAR is encompassed by a polypeptide that furthercomprises a signaling domain selected from a DAP10 signaling domain (SEQID NO: 29 and SEQ ID NO: 135), or a DAP12 signaling domain (SEQ ID NO:30 and SEQ ID NO: 136). In some cases, the EGFRvIII-specific CAR isencompassed by a polypeptide that further comprises a signaling domainselected from a DAP10 signaling domain (SEQ ID NO: 29 and SEQ ID NO:135), or a DAP12 signaling domain (SEQ ID NO: 30 and SEQ ID NO: 136). Insome instances, the EGFRvIII-specific CAR further comprises a stalkregion and a stalk extension region as disclosed herein. For example,the EGFRvIII-specific CAR can further comprise a spacer wherein thespacer comprises a stalk region comprising a CD8α hinge region, andstalk extension region(s), s′-n, wherein n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, wherein each stalkextension region being homologous to a CD8α hinge region except forlacking a dimerization site.

In some embodiments, the EGFRvIII-specific CAR described hereincomprises EGFRvIII-specific chimeric antigen receptor with CD8-1× spacer(clone 139scFv-CD8α-4-1BB-CD3ζ) comprising a peptide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 232 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 231. In some embodiments, theEGFRvIII-specific CAR described herein comprises EGFRvIII-specificchimeric antigen receptor with CD8-1× spacer (cloneMR1scFv-CD8α-4-1BB-CD3ζ) comprising a peptide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 234 or a peptide sequenceencoded by a nucleotide sequence with at least 80% homology to thesequence shown in SEQ ID NO: 233.

In some embodiments, the EGFRvIII-specific CAR described hereincomprises EGFRvIII-specific chimeric antigen receptor with CD8-1× spacer(clone MR1-1scFv-CD8α-4-1BB-CD3ζ) comprising a peptide sequence with atleast 80% homology to the sequence shown in SEQ ID NO: 236 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 235. In some embodiments, theEGFRvIII-specific CAR described herein comprises EGFRvIII specificchimeric antigen receptor with CD8-2× spacer comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:242 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 241. In someembodiments, the EGFRvIII-specific CAR described herein comprisesEGFRvIII specific chimeric antigen receptor with CD8-3× spacercomprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 244 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:243. In some embodiments, the EGFRvIII-specific CAR described hereincomprises EGFRvIII specific chimeric antigen receptor with CD8-4× spacercomprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 246 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:245.

In some embodiments, the EGFRvIII-specific CAR described hereincomprises EGFRvIII-specific chimeric antigen receptor with CD8-1× spacer(huMR1-1-CD8α-4-1BB-CD3ζ) comprising a peptide sequence with at least80% homology to the sequence shown in SEQ ID NO: 238 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 237. In some embodiments, theEGFRvIII-specific CAR described herein comprises EGFRvIII-specificchimeric antigen receptor with CD8-3× spacer comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:248 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 247. In someembodiments, the EGFRvIII-specific CAR described herein comprisesEGFRvIII-specific chimeric antigen receptor with CD8-4× spacercomprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 250 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:249.

In some embodiments, the EGFRvIII-specific CAR described hereincomprises EGFRvIII-specific chimeric antigen receptor with CD8-1× spacer(huMR1-2-CD8α-4-1BB-CD3ζ) comprising a peptide sequence with at least80% homology to the sequence shown in SEQ ID NO: 240 or a peptidesequence encoded by a nucleotide sequence with at least 80% homology tothe sequence shown in SEQ ID NO: 239. In some embodiments, theEGFRvIII-specific CAR described herein comprises EGFRvIII-specificchimeric antigen receptor with CD8-3× spacer comprising a peptidesequence with at least 80% homology to the sequence shown in SEQ ID NO:252 or a peptide sequence encoded by a nucleotide sequence with at least80% homology to the sequence shown in SEQ ID NO: 251. In someembodiments, the EGFRvIII-specific CAR described herein comprisesEGFRvIII-specific chimeric antigen receptor with CD8-4× spacercomprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 254 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:253.

Modified Effector Cells

In some embodiments, modified effector cells expressing polypeptides aredescribed herein, including antigen binding polypeptides such as CARsdescribed herein. In some embodiments, the modified effector cells aremodified immune cells that comprise T cells and/or natural killer cells.T cells or T lymphocytes are a subtype of white blood cells that areinvolved in cell-mediated immunity. Exemplary T cells include T helpercells, cytotoxic T cells, TH17 cells, stem memory T cells (T_(SCM)),naïve T cells, memory T cells, effector T cells, regulatory T cells, ornatural killer T cells.

T helper cells (TH cells) assist other white blood cells in immunologicprocesses, including maturation of B cells into plasma cells and memoryB cells, and activation of cytotoxic T cells and macrophages. In someinstances, TH cells are known as CD4+ T cells due to expression of theCD4 glycoprotein on the cell surfaces. Helper T cells become activatedwhen they are presented with peptide antigens by MHC class II molecules,which are expressed on the surface of antigen-presenting cells (APCs).Once activated, they divide rapidly and secrete small proteins calledcytokines that regulate or assist in the active immune response. Thesecells can differentiate into one of several subtypes, including TH1,TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines tofacilitate different types of immune responses. Signaling from the APCdirects T cells into particular subtypes.

Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells andtumor cells, and are also implicated in transplant rejection. Thesecells are also known as CD8+ T cells since they express the CD8glycoprotein at their surfaces. These cells recognize their targets bybinding to antigen associated with MHC class I molecules, which arepresent on the surface of all nucleated cells. Through IL-10, adenosine,and other molecules secreted by regulatory T cells, the CD8+ cells canbe inactivated to an anergic state, which prevents autoimmune diseases.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise subtypes: stem memory T cells (T_(SCM)), centralmemory T cells (T_(CM) cells) and two types of effector memory T cells(T_(EM) cells and T_(EMR)A cells). Memory cells may be either CD4+ orCD8+. Memory T cells may express the cell surface proteins CD45RO,CD45RA and/or CCR7.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,play a role in the maintenance of immunological tolerance. Their majorrole is to shut down T cell-mediated immunity toward the end of animmune reaction and to suppress autoreactive T cells that escaped theprocess of negative selection in the thymus.

Natural killer T cells (NKT cells) bridge the adaptive immune systemwith the innate immune system. Unlike conventional T cells thatrecognize peptide antigens presented by major histocompatibility complex(MHC) molecules, NKT cells recognize glycolipid antigen presented by amolecule called CD1d. Once activated, these cells can perform functionsascribed to both Th and Tc cells (i.e., cytokine production and releaseof cytolytic/cell killing molecules). They are also able to recognizeand eliminate some tumor cells and cells infected with herpes viruses.

Natural killer (NK) cells are a type of cytotoxic lymphocyte of theinnate immune system. In some instances, NK cells provide a first linedefense against viral infections and/or tumor formation. NK cells candetect MHC presented on infected or cancerous cells, triggering cytokinerelease, and subsequently induce lysis and apoptosis. NK cells canfurther detect stressed cells in the absence of antibodies and/or MHC,thereby allowing a rapid immune response.

Engineered T-Cell Receptor (TCR)

In some embodiments, a polypeptide described herein comprises anengineered T-cell receptor. The T cell receptor (TCR) is composed of twochains (αβ or γδ) that pair on the surface of the T cell to form aheterodimeric receptor. In some instances, the αβ TCR is expressed onmost T cells in the body and is known to be involved in the recognitionof specific MHC-restricted antigens. Each α and β chain are composed oftwo domains: a constant domain (C) which anchors the protein to the cellmembrane and is associated with invariant subunits of the CD3 signalingapparatus; and a variable domain (V) that confers antigen recognitionthrough six loops, referred to as complementarity determining regions(CDRs). In some instances, each of the V domains comprises three CDRs;e.g., CDR1, CDR2 and CDR3 with CDR3 as the hypervariable region. TheseCDRs interact with a complex formed between an antigenic peptide boundto a protein encoded by the major histocompatibility complex (pepMHC)(e.g., HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1,HLA-DRA, or HLA-DRB1 complex). In some instances, the constant domainfurther comprises a joining region that connects the constant domain tothe variable domain. In some cases, the beta chain further comprises ashort diversity region which makes up part of the joining region.

In some cases, such TCR are reactive to specific tumor antigen, e.g.NY-ESO, Titin, MART-1, HPV, HBV, MAGE-A4, MAGE-A10, MAGE A3/A6, gp100,MAGE-A1, or PRAME In other cases, such TCR are reactive to specificneoantigens expressed within a patient's tumor (i.e. patient-specific,somatic, non-synonymous mutations expressed by tumors). In some cases,engineered TCRs can be affinity-enhanced.

In some embodiments, a TCR is described using the InternationalImmunogenetics (IMGT) TCR nomenclature, and links to the IMGT publicdatabase of TCR sequences. For example, there can be several types ofalpha chain variable (Vα) regions and several types of beta chainvariable (Vβ) regions distinguished by their framework, CDR1, CDR2, andCDR3 sequences. As such, a Vα type can be referred to in IMGTnomenclature by a unique TRAV number. For example, “TRAV21” defines aTCR Vα region having unique framework and CDR1 and CDR2 sequences, and aCDR3 sequence which is partly defined by an amino acid sequence which ispreserved from TCR to TCR but which also includes an amino acid sequencewhich varies from TCR to TCR. Similarly, “TRBV5-1” defines a TCR Vβregion having unique framework and CDR1 and CDR2 sequences, but withonly a partly defined CDR3 sequence.

In some cases, the beta chain diversity region is referred to in IMGTnomenclature by the abbreviation TRBD.

In some instances, the unique sequences defined by the IMGT nomenclatureare widely known and accessible to those working in the TCR field. Forexample, they can be found in the IMGT public database and in “T cellReceptor Factsbook,” (2001) LeFranc and LeFranc, Academic Press, ISBN0-12-441352-8.

In some embodiments, an as heterodimeric TCR is, for example,transfected as full length chains having both cytoplasmic andtransmembrane domains. In some cases, the TCRs contain an introduceddisulfide bond between residues of the respective constant domains, asdescribed, for example, in WO 2006/000830.

In some instances, TCRs described herein are in single chain format, forexample see WO 2004/033685. Single chain formats include αβ TCRpolypeptides of the Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vβ, Vα-L-Vβ-Cβ,Vα-Cα-L-Vβ-Cβ types, wherein Vα and Vβ are TCR α and β variable regionsrespectively, Cα and Cβ are TCR α and β constant regions respectively,and L is a linker sequence. In certain embodiments single chain TCRs ofthe invention may have an introduced disulfide bond between residues ofthe respective constant domains, as described in WO 2004/033685.

The TCR described herein may be associated with a detectable label, atherapeutic agent or a PK modifying moiety.

Exemplary detectable labels for diagnostic purposes include, but are notlimited to, fluorescent labels, radiolabels, enzymes, nucleic acidprobes and contrast reagents.

In some cases, each chain of TCR disclosed herein, for example αβ or γδ,comprises a modified spacer region connecting constant region of a TCRchain to transmembrane region.

In some cases, a spacer region of each chain of TCR disclosed hereincomprises 1) a stalk region and 2) stalk extension region(s) (s′-n,wherein n=0, 1, 2, 3 or more) adjacent to said stalk region.Illustrative embodiments are described in FIG. 1A and FIG. 1B. In someembodiments, each chain of TCR disclosed herein incorporates a spacerthat comprises a stalk region (s) and a stalk extension region (s′-n,wherein n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20).

As described herein, a spacer region can comprise a stalk region andstalk extension region(s). In some instances, the stalk region comprisesthe extracellular hinge region from TCRα or TCRβ chain or the stalkregion comprises a sequence with at least 80% homology to theextracellular hinge region from TCRα or TCRβ chain. For example, thestalk region can comprise a sequence with at least 80%, 85%, 90%, 95%,or greater than 95% homology to the hinge region of the extracellularregion of TCRα or TCRβ chain. In alternative instances, the stalk regioncomprises any portion of extracellular region of TCRα or TCRβ constantregion with at least 80% homology to the extracellular region of TCRα orTCRβ constant region respectively. For example, the stalk region cancomprise a sequence with at least 80%, 85%, 90%, 95%, or greater than95% homology to the any portion of extracellular region of TCRα or TCRβconstant region.

TCR chain heterodimers are formed by inter-chain disulfide bonds inextracellular hinge region of α and β chains. In some embodiments, thestalk region comprises a dimerization site. A dimerization site cancomprise a disulfide bond formation site. A dimerization site cancomprise cysteine residue(s). A stalk region can be capable of forming adisulfide bond. Such a disulfide bond can be formed at a disulfide bondforming site or a dimerization site. In some examples, the dimerizationoccurs between α and β chains of TCR.

In some embodiments, a stalk extension region is used. In someembodiments, a stalk extension region is used to link the stalk regionto the transmembrane region TCR α and β chains. In additionalembodiments, a stalk extension region is used to link the stalk regionto constant region of TCR α and β chains. In another embodiment, thestalk region and the stalk extension region(s) can be connected via alinker.

In some instances, the stalk extension domain comprises a sequence thatis partially homologous to the stalk region. In some instances, each ofthe stalk extension region comprises a sequence that is homologous tothe stalk region, except that the stalk extension region lacks thedimerization site of the stalk region. In some cases, each of the stalkextension region comprises a sequence identical to the stalk region. Inother cases, each of the stalk extension regions comprise a sequenceidentical to the stalk region with at least one amino acid residuesubstitution relative to the stalk region. In some cases, each of thestalk extension region is not capable of forming a disulfide bond or isnot capable of dimerization with a homologous stalk extension region.

In other embodiments, one stalk extension region can be connected toanother stalk extension region via a linker. Examples of such linkerscan include glycine-serine rich linkers.

In some embodiments, addition of stalk extension region(s) preventsmispairing of transgenic TCR α and β chains with native TCR α and βchains expressed by T cells that are genetically modified.

In some embodiments, the TCR described herein comprises TCRα chainconstant region comprising a peptide sequence with at least 80% homologyto the sequence shown in SEQ ID NO: 69 or a peptide sequence encoded bya nucleotide sequence with at least 80% homology to the sequence shownin SEQ ID NO: 163. In some embodiments, the TCR described hereincomprises TCRβ1 chain constant region comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 83 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 177. In some embodiments,the TCR described herein comprises TCRβ2 chain constant regioncomprising a peptide sequence with at least 80% homology to the sequenceshown in SEQ ID NO: 97 or a peptide sequence encoded by a nucleotidesequence with at least 80% homology to the sequence shown in SEQ ID NO:191. In some embodiments, the TCR described herein comprises TCRγ1 chainconstant region comprising a peptide sequence with at least 80% homologyto the sequence shown in SEQ ID NO: 99 or a peptide sequence encoded bya nucleotide sequence with at least 80% homology to the sequence shownin SEQ ID NO: 193. In some embodiments, the TCR described hereincomprises TCRγ2 chain constant region comprising a peptide sequence withat least 80% homology to the sequence shown in SEQ ID NO: 102 or apeptide sequence encoded by a nucleotide sequence with at least 80%homology to the sequence shown in SEQ ID NO: 196. In some embodiments,the TCR described herein comprises TCRδ chain constant region comprisinga peptide sequence with at least 80% homology to the sequence shown inSEQ ID NO: 104 or a peptide sequence encoded by a nucleotide sequencewith at least 80% homology to the sequence shown in SEQ ID NO: 198.

In some aspects of the embodiments disclosed herein, a stalk region or astalk extension region of a polypeptide described herein comprises apeptide sequence with at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 25%, 26%, 27%, 28%,29%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% orgreater identity to the extracellular region of human TCRα chainconstant region sequence shown in SEQ ID NO: 70. In some cases, a stalkregion or a stalk extension region of a polypeptide described hereincomprises a peptide sequence encoded by a nucleotide sequence with atleast about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 50%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to the humanTCRα chain constant region nucleotide sequence shown in SEQ ID NO: 164.In some cases, a stalk extension region comprises a sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater identity to thesequence shown in SEQ ID NO: 72 or SEQ ID NO: 73 or a peptide sequenceencoded by a nucleotide sequence with at least 65%, 70%, 75%, 80%, 85%,90%, 95%, 99% or greater identity to the sequence shown in SEQ ID NO:166 or SEQ ID NO: 167. In some examples, a stalk region and stalkextension region can together comprise a peptide sequence with at least65%, 70%, 75%, 80%, 85%, 90%, 95% or greater identity to the sequenceshown in SEQ ID NO: 77, SEQ ID NO: 78 or SEQ ID NO: 79, or a peptidesequence encoded by a nucleotide sequence with at least 65%, 70%, 75%,80%, 85%, 90%, 95%, 99% or greater identity to the sequence shown in SEQID NO: 171, SEQ ID NO: 172 or SEQ ID NO: 173. In some examples, a stalkregion and stalk extension region can together comprise a peptidesequence with at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or greateridentity to the sequence shown in SEQ ID NO: 80, SEQ ID NO: 81 or SEQ IDNO: 82, or a peptide sequence encoded by a nucleotide sequence with atleast 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater identity to thesequence shown in SEQ ID NO: 174, SEQ ID NO: 175 or SEQ ID NO: 176.

In some embodiment, the stalk region and the stalk extension region(s)can be connected via a linker, such as whitlow linker (SEQ ID NO: 8 andSEQ ID NO: 114), GSG linker (SEQ ID NO: 9 and SEQ ID NO: 115), SGSGlinker (SEQ ID NO: 10) or (G4S)3 linker (SEQ ID NO: 11 and SEQ ID NO:117). In one embodiment, the TCRα hinge domain connected to (G4S)3linker (SEQ ID NO: 11) can comprise a peptide sequence shown in SEQ IDNO: 74 or a peptide sequence encoded by a nucleotide sequence shown inSEQ ID NO: 168. In one embodiment, the TCRβ hinge domain connected to(G4S)3 linker (SEQ ID NO: 11) can comprise a peptide sequence shown inSEQ ID NO: 75 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 169. In yet another embodiment, the TCRβ hingedomain connected to whitlow linker can comprise a peptide sequence shownin SEQ ID NO: 76 or a peptide sequence encoded by a nucleotide sequenceshown in SEQ ID NO: 170.

In some aspects of the embodiments disclosed herein, the extracellularregion of human TCRα chain constant region described herein comprises apeptide sequence with at least 80% or greater identity to a peptidesequence shown in SEQ ID NO: 70. In some cases, the extracellular regionof human TCRβ 1 chain constant region described herein comprises apeptide sequence encoded by a nucleotide sequence with at least about80% or greater identity to the human TCRβ1 chain constant regionnucleotide sequence shown in SEQ ID NO: 164. In some aspects of theembodiments disclosed herein, the extracellular region of human TCRβ1chain constant region described herein comprises a peptide sequence withat least 80% or greater identity to a peptide sequence shown in SEQ IDNO: 84. In some cases, the extracellular region of human TCRβ1 chainconstant region described herein comprises a peptide sequence encoded bya nucleotide sequence with at least about 80% or greater identity to thehuman TCRβ 1 chain constant region nucleotide sequence shown in SEQ IDNO: 178. In some aspects of the embodiments disclosed herein, theextracellular region of human TCRβ2 chain constant region describedherein comprises a peptide sequence with at least 80% or greateridentity to a peptide sequence shown in SEQ ID NO: 98. In some cases,the extracellular region of human TCRβ2 chain constant region describedherein comprises a peptide sequence encoded by a nucleotide sequencewith at least about 80% or greater identity to the human TCRβ1 chainconstant region nucleotide sequence shown in SEQ ID NO: 192. In someaspects of the embodiments disclosed herein, the extracellular region ofhuman TCRγ1 chain constant region described herein comprises a peptidesequence with at least 80% or greater identity to a peptide sequenceshown in SEQ ID NO: 100. In some cases, the extracellular region ofhuman TCR 71 chain constant region described herein comprises a peptidesequence encoded by a nucleotide sequence with at least about 80% orgreater identity to the human TCRβ 1 chain constant region nucleotidesequence shown in SEQ ID NO: 194. In some aspects of the embodimentsdisclosed herein, the extracellular region of human TCRγ2 chain constantregion described herein comprises a peptide sequence with at least 80%or greater identity to a peptide sequence shown in SEQ ID NO: 103. Insome cases, the extracellular region of human TCR 72 chain constantregion described herein comprises a peptide sequence encoded by anucleotide sequence with at least about 80% or greater identity to thehuman TCRβ1 chain constant region nucleotide sequence shown in SEQ IDNO: 197. In some aspects of the embodiments disclosed herein, theextracellular region of human TCRS chain constant region describedherein comprises a peptide sequence with at least 80% or greateridentity to a peptide sequence shown in SEQ ID NO: 105. In some cases,the extracellular region of human TCRS chain constant region describedherein comprises a peptide sequence encoded by a nucleotide sequencewith at least about 80% or greater identity to the human TCRβ 1 chainconstant region nucleotide sequence shown in SEQ ID NO: 199.

Modified Effector Cell Doses

In some embodiments, an amount of modified effector cells isadministered to a subject in need thereof and the amount is determinedbased on the efficacy and the potential of inducing acytokine-associated toxicity. In some cases, an amount of modifiedimmune effector cells comprises about 10² to about 10⁹ modified immuneeffector cells/kg. In some cases, an amount of modified immune effectorcells comprises about 10³ to about 10⁹ modified immune effectorcells/kg. In some cases, an amount of modified effector cells comprisesabout 10⁴ to about 10⁹ modified effector cells/kg. In some cases, anamount of modified effector cells comprises about 10⁵ to about 10⁹modified effector cells/kg. In some cases, an amount of modifiedeffector cells comprises about 10⁵ to about 10⁸ modified effectorcells/kg. In some cases, an amount of modified effector cells comprisesabout 10⁵ to about 10⁷ modified effector cells/kg. In some cases, anamount of modified effector cells comprises about 10⁶ to about 10⁹modified effector cells/kg. In some cases, an amount of modifiedeffector cells comprises about 10⁶ to about 10⁸ modified effectorcells/kg. In some cases, an amount of modified effector cells comprisesabout 10⁷ to about 10⁹ modified effector cells/kg. In some cases, anamount of modified effector cells comprises about 10⁵ to about 10⁶modified effector cells/kg. In some cases, an amount of modifiedeffector cells comprises about 10⁶ to about 10⁷ modified effectorcells/kg. In some cases, an amount of modified effector cells comprisesabout 10⁷ to about 10⁸ modified effector cells/kg. In some cases, anamount of modified effector cells comprises about 10⁸ to about 10⁹modified effector cells/kg. In some instances, an amount of modifiedeffector cells comprises about 10⁹ modified effector cells/kg. In someinstances, an amount of modified effector cells comprises about 10⁸modified effector cells/kg. In some instances, an amount of modifiedeffector cells comprises about 10⁷ modified effector cells/kg. In someinstances, an amount of modified effector cells comprises about 10⁶modified effector cells/kg. In some instances, an amount of modifiedeffector cells comprises about 10⁵ modified effector cells/kg. In someinstances, an amount of modified effector cells comprises about 10⁴modified effector cells/kg.

In some embodiments, the modified effector cells expressing apolypeptide described herein, are modified T cells. In some instances,the modified T cells are CAR-T cells. In some cases, an amount of CAR-Tcells comprises about 10² to about 10⁹ CAR-T cells/kg. In some cases, anamount of CAR-T cells comprises about 10³ to about 10⁹ CAR-T cells/kg.In some cases, an amount of CAR-T cells comprises about 10⁴ to about 10⁹CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about10⁵ to about 10⁸ CAR-T cells/kg. In some cases, an amount of CAR-T cellscomprises about 10⁵ to about 10⁷ CAR-T cells/kg. In some cases, anamount of CAR-T cells comprises about 10⁶ to about 10⁹ CAR-T cells/kg.In some cases, an amount of CAR-T cells comprises about 10⁶ to about 10⁸CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about10⁷ to about 10⁹ CAR-T cells/kg. In some cases, an amount of CAR-T cellscomprises about 10⁵ to about 10⁶ CAR-T cells/kg. In some cases, anamount of CAR-T cells comprises about 10⁶ to about 10⁷ CAR-T cells/kg.In some cases, an amount of CAR-T cells comprises about 10⁷ to about 10⁸CAR-T cells/kg. In some cases, an amount of CAR-T cells comprises about10⁸ to about 10⁹ CAR-T cells/kg. In some instances, an amount of CAR-Tcells comprises about 10⁹ CAR-T cells/kg. In some instances, an amountof CAR-T cells comprises about 10⁸ CAR-T cells/kg. In some instances, anamount of CAR-T cells comprises about 10⁷ CAR-T cells/kg. In someinstances, an amount of CAR-T cells comprises about 10⁶ CAR-T cells/kg.In some instances, an amount of CAR-T cells comprises about 10⁵ CAR-Tcells/kg. In some instances, an amount of CAR-T cells comprises about10⁴ CAR-T cells/kg. In some instances, an amount of CAR-T cellscomprises about 10³ CAR-T cells/kg. In some instances, an amount ofCAR-T cells comprises about 10² CAR-T cells/kg.

In some embodiments, the CAR-T cells are CD19-specific CAR-T cells. Insome cases, an amount of CD19-specific CAR-T cells comprises about 10²to about 10⁹ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10³ to about 10⁹ CAR-T cells/kg. In somecases, an amount of CD19-specific CAR-T cells comprises about 10⁴ toabout 10⁹ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10⁵ to about 10⁸ CAR-T cells/kg. In somecases, an amount of CD19-specific CAR-T cells comprises about 10⁵ toabout 10⁷ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10⁶ to about 10⁹ CAR-T cells/kg. In somecases, an amount of CD19-specific CAR-T cells comprises about 10⁶ toabout 10⁸ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10⁷ to about 10⁹ CAR-T cells/kg. In somecases, an amount of CD19-specific CAR-T cells comprises about 10⁵ toabout 10⁶ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10⁶ to about 10⁷ CAR-T cells/kg. In somecases, an amount of CD19-specific CAR-T cells comprises about 10⁷ toabout 10⁸ CAR-T cells/kg. In some cases, an amount of CD19-specificCAR-T cells comprises about 10⁸ to about 10⁹ CAR-T cells/kg. In someinstances, an amount of CD19-specific CAR-T cells comprises about 10⁹CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-Tcells comprises about 10⁸ CAR-T cells/kg. In some instances, an amountof CD19-specific CAR-T cells comprises about 10⁷ CAR-T cells/kg. In someinstances, an amount of CD19-specific CAR-T cells comprises about 10⁶CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-Tcells comprises about 10⁵ CAR-T cells/kg. In some instances, an amountof CD19-specific CAR-T cells comprises about 10⁴ CAR-T cells/kg. In someinstances, an amount of CD19-specific CAR-T cells comprises about 10³CAR-T cells/kg. In some instances, an amount of CD19-specific CAR-Tcells comprises about 10² CAR-T cells/kg.

In some embodiments, a polypeptide described herein is expressed inmodified T cells which are engineered TCR T-cells. In some cases, anamount of engineered TCR T-cells comprises about 10² to about 10⁹ TCRcells/kg. In some cases, an amount of engineered TCR T-cells comprisesabout 10³ to about 10⁹ TCR cells/kg. In some cases, an amount ofengineered TCR T-cells comprises about 10⁴ to about 10⁹ TCR cells/kg. Insome cases, an amount of engineered TCR T-cells comprises about 10⁵ toabout 10⁹ TCR cells/kg. In some cases, an amount of engineered TCR cellscomprises about 10⁵ to about 10⁸ TCR cells/kg. In some cases, an amountof engineered TCR cells comprises about 10⁵ to about 10⁷ TCR cells/kg.In some cases, an amount of engineered TCR cells comprises about 10⁶ toabout 10⁹ TCR cells/kg. In some cases, an amount of engineered TCR cellscomprises about 10⁶ to about 10⁸ TCR cells/kg. In some cases, an amountof engineered TCR cells comprises about 10⁷ to about 10⁹ TCR cells/kg.In some cases, an amount of engineered TCR cells comprises about 10⁵ toabout 10⁶ TCR cells/kg. In some cases, an amount of engineered TCR cellscomprises about 10⁶ to about 10⁷ TCR cells/kg. In some cases, an amountof engineered TCR cells comprises about 10⁷ to about 10⁸ TCR cells/kg.In some cases, an amount of engineered TCR cells comprises about 10⁸ toabout 10⁹ TCR cells/kg. In some instances, an amount of engineered TCRcells comprises about 10⁹ TCR cells/kg. In some instances, an amount ofengineered TCR cells comprises about 10⁸ TCR cells/kg. In someinstances, an amount of engineered TCR cells comprises about 10⁷ TCRcells/kg. In some instances, an amount of engineered TCR cells comprisesabout 10⁶ TCR cells/kg. In some instances, an amount of engineered TCRcells comprises about 10⁵ TCR cells/kg. In some instances, an amount ofengineered TCR cells comprises about 10⁴ TCR cells/kg. In someinstances, an amount of engineered TCR cells comprises about 10³ TCRcells/kg. In some instances, an amount of engineered TCR cells comprisesabout 10² TCR cells/kg.

Indications

In some embodiments, disclosed herein are methods of administering amodified effector cell comprising a polypeptide described herein to asubject having a disorder, for instance a cancer. In some cases, thecancer is a cancer associated with an expression of CD19, CD20, CD33,CD44, BCMA, CD123, EGFRvIII, α-Folate receptor, CAIX, CD30, ROR1, CEA,EGP-2, EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2,KDR, EDB-F, mesothelin, GPC3, CSPG4, HER1/HER3, HER2, CD44v6, CD44v7/v8,CD20, CD174, CD138, L1-CAM, FAP, c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2,CLL-1, CD22, EGFR, Folate receptor α, Mucins such as MUC-1 or MUC-16,MAGE-A1, h5T4, PSMA, CSPG4, TAG-72 or VEGF-R2.

In some embodiments, disclosed herein are methods of administering apolynucleotide, polypeptide or a modified effector cell encoding apolynucleotide described herein, to a subject having a cancer associatedwith an overexpression of CD19. In some embodiments, disclosed hereinare methods of administering a modified effector cell to a subjecthaving a cancer associated with an overexpression of CD33. In someembodiments, disclosed herein are methods of administering a modifiedeffector cell to a subject having a cancer associated with anoverexpression of CD44, CD19, BCMA, CD123, EGFRvIII, α-Folate receptor,CAIX, CD30, ROR1, CEA, EGP-2, EGP-40, HER2, HER3, Folate-bindingProtein, GD2, GD3, IL-13R-a2, KDR, EDB-F, mesothelin, GPC3, CSPG4,HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP,c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2, CLL-1, CD22, EGFR, Mucins suchas MUC-1 or MUC-16, MAGE-A1, h5T4, PSMA, TAG-72 or VEGF-R2. In somecases, the cancer is a metastatic cancer. In other cases, the cancer isa relapsed or refractory cancer.

In some cases, a cancer is a solid tumor or a hematologic malignancy. Insome instances, the cancer is a solid tumor. In other instances, thecancer is a hematologic malignancy. In some cases, the cancer is ametastatic cancer. In some cases, the cancer is a relapsed or refractorycancer.

In some instances, the cancer is a solid tumor. Exemplary solid tumorsinclude, but are not limited to, anal cancer; appendix cancer; bile ductcancer (i.e., cholangiocarcinoma); bladder cancer; brain tumor; breastcancer; cervical cancer; colon cancer; cancer of Unknown Primary (CUP);esophageal cancer; eye cancer; fallopian tube cancer;gastroenterological cancer; kidney cancer; liver cancer; lung cancer;medulloblastoma; melanoma; oral cancer; ovarian cancer; pancreaticcancer; parathyroid disease; penile cancer; pituitary tumor; prostatecancer; rectal cancer; skin cancer; stomach cancer; testicular cancer;throat cancer; thyroid cancer; uterine cancer; vaginal cancer; or vulvarcancer.

In some instances, the cancer is a hematologic malignancy. In somecases, a hematologic malignancy comprises a lymphoma, a leukemia, amyeloma, or a B-cell malignancy. In some cases, a hematologic malignancycomprises a lymphoma, a leukemia or a myeloma. In some instances,exemplary hematologic malignancies include chronic lymphocytic leukemia(CLL), small lymphocytic lymphoma (SLL), high risk CLL, non-CLL/SLLlymphoma, prolymphocytic leukemia (PLL), follicular lymphoma (FL),diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL),Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginalzone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt'slymphoma, non-Burkitt high grade B cell lymphoma, primary mediastinalB-cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursorB-lymphoblastic lymphoma, B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cellmyeloma, plasmacytoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma, orlymphomatoid granulomatosis. In some embodiments, the hematologicmalignancy comprises a myeloid leukemia. In some embodiments, thehematologic malignancy comprises acute myeloid leukemia (AML) or chronicmyeloid leukemia (CML).

In some instances, disclosed herein are methods of administering to asubject having a hematologic malignancy selected from chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), high riskCLL, non-CLL/SLL lymphoma, prolymphocytic leukemia (PLL), follicularlymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle celllymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma,extranodal marginal zone B cell lymphoma, nodal marginal zone B celllymphoma, Burkitt's lymphoma, non-Burkitt high grade B cell lymphoma,primary mediastinal B-cell lymphoma (PMBL), immunoblastic large celllymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma,plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B celllymphoma, intravascular large B cell lymphoma, primary effusionlymphoma, or lymphomatoid granulomatosis a modified effector celldescribed herein. In some instances, disclosed herein are methods ofadministering to a subject having a hematologic malignancy selected fromAML or CML a modified effector cell to the subject.

Viral Based Delivery Systems

Certain embodiments disclosed herein also provide delivery systems, suchas viral-based systems, in which a nucleic acid encoding a polypeptidedescribed herein is inserted. Representative viral expression vectorsinclude, but are not limited to, adeno-associated viral vectors,adenovirus-based vectors (e.g., the adenovirus-based Per.C6 systemavailable from Crucell, Inc. (Leiden, The Netherlands)),lentivirus-based vectors (e.g., the lentiviral-based pLPI from LifeTechnologies (Carlsbad, Calif.)), retroviral vectors (e.g., the pFB-ERVplus pCFB-EGSH), and herpes virus-based vectors. In an embodiment, theviral vector is a lentivirus vector. Vectors derived from retrovirusessuch as the lentivirus are suitable tools to achieve long-term genetransfer since they allow long-term, stable integration of a transgeneand its propagation in daughter cells. Lentiviral vectors have the addedadvantage over vectors derived from onco-retroviruses such as murineleukemia viruses in that they can transduce non-proliferating cells,such as hepatocytes. They also have the added advantage of lowimmunogenicity. In an additional embodiment, the viral vector is anadeno-associated viral vector. In a further embodiment, the viral vectoris a retroviral vector. In general, and in embodiments, a suitablevector contains an origin of replication functional in at least oneorganism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers, (e.g., WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Additional suitable vectors include integrating expression vectors,which may randomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system fromInvitrogen (Carlsbad, Calif.) (e.g., pcDNA™ 5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Invitrogen (Carlsbad,Calif.), and pCI or pFN10A (ACT) FLEXI™ from Promega (Madison, Wis.).Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region30-110 bp upstream of the start site, although a number of promotershave recently been shown to contain functional elements downstream ofthe start site as well. The spacing between promoter elements frequentlyis flexible, so that promoter function is preserved when elements areinverted or moved relative to one another. In the thymidine kinase (tk)promoter, the spacing between promoter elements can be increased to 50bp apart before activity begins to decline. Depending on the promoter,it appears that individual elements can function either cooperatively orindependently to activate transcription.

One example of a suitable promoter is the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.

Another example of a suitable promoter is human elongation growth factor1 alpha 1 (hEF1a1). In embodiments, the vector construct comprising theCARs and/or TCRs described herein comprises hEF1a1 functional variants.

However, other constitutive promoter sequences may also be used,including, but not limited to the simian virus 40 (SV40) early promoter,mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV)long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemiavirus promoter, an Epstein-Barr virus immediate early promoter, a Roussarcoma virus promoter, as well as human gene promoters such as, but notlimited to, the actin promoter, the myosin promoter, the hemoglobinpromoter, and the creatine kinase promoter. Cell type specific, forexample T cell specific, promoters can also be used. Further, thedisclosed embodiments should not be limited to the use of constitutivepromoters. Inducible promoters are also contemplated as part of one ormore embodiments disclosed herein. The use of an inducible promoterprovides a molecular switch capable of turning on expression of thepolynucleotide sequence which it is operatively linked when suchexpression is desired, or turning off the expression when expression isnot desired. Examples of inducible promoters include, but are notlimited to a metallothionine promoter, a glucocorticoid promoter, aprogesterone promoter, and a tetracycline promoter. In one aspect, theinducible promoter can be a gene switch ligand inducible promoter. Insome cases, an inducible promoter can be a small moleculeligand-inducible two polypeptide ecdysone receptor-based gene switch,such as RHEOSWITCH® gene switch.

In order to assess the expression of a CAR or TCR polypeptide orportions thereof, the expression vector to be introduced into a cell canalso contain either a selectable marker gene or a reporter gene or bothto facilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In other aspects, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, for example, antibiotic-resistance genes,such as neomycin resistance gene (neo) and ampicillin resistance geneand the like. In some embodiments, a truncated epidermal growth factorreceptor (HER1t) tag may be used as a selectable marker gene.

Reporter genes can be used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assayed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include genes encoding luciferase,beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene (e.g.,Ui-Tei et al., FEBS Letters 479: 79-82 (2000)). Suitable expressionsystems are well known and may be prepared using known techniques orobtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter geneis identified as the promoter. Such promoter regions may be linked to areporter gene and used to evaluate agents for the ability to modulatepromoter-driven transcription.

In some embodiments, the vectors comprise a hEF1a1 promoter to driveexpression of transgenes, a bovine growth hormone polyA sequence toenhance transcription, a woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE), as well as LTR sequences derived from thepFUGW plasmid.

Methods of introducing and expressing genes into a cell are known in theart. In the context of an expression vector, the vector can be readilyintroduced into a host cell, e.g., mammalian, bacterial, yeast, orinsect cell by any method in the art. For example, the expression vectorcan be transferred into a host cell by physical, chemical, or biologicalmeans.

Physical methods for introducing a polynucleotide into a host cellinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells comprising vectors and/or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York(2001)). In embodiments, a method for the introduction of apolynucleotide into a host cell is calcium phosphate transfection orpolyethylenimine (PEI) Transfection.

Biological methods for introducing a polynucleotide encoding apolypeptide of interest into a host cell include the use of DNA and RNAvectors. Viral vectors, and especially retroviral vectors, have becomethe most widely used method for inserting genes into mammalian, e.g.,human cells. Other viral vectors can be derived from lentivirus,poxviruses, herpes simplex virus I, adenoviruses and adeno-associatedviruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

In certain embodiments, an exemplary delivery vehicle is a liposome. Theuse of lipid formulations is contemplated for the introduction of thenucleic acids into a host cell (in vitro, ex vivo or in vivo). Inanother aspect, the nucleic acid may be associated with a lipid. Thenucleic acid associated with a lipid may be encapsulated in the aqueousinterior of a liposome, interspersed within the lipid bilayer of aliposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the oligonucleotide, entrapped ina liposome, complexed with a liposome, dispersed in a solutioncontaining a lipid, mixed with a lipid, combined with a lipid, containedas a suspension in a lipid, contained or complexed with a micelle, orotherwise associated with a lipid. Lipid, lipid/DNA or lipid/expressionvector associated compositions are not limited to any particularstructure in solution. For example, they may be present in a bilayerstructure, as micelles, or with a “collapsed” structure. They may alsosimply be interspersed in a solution, possibly forming aggregates thatare not uniform in size or shape. Lipids are fatty substances which maybe naturally occurring or synthetic lipids. For example, lipids includethe fatty droplets that naturally occur in the cytoplasm as well as theclass of compounds which contain long-chain aliphatic hydrocarbons andtheir derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,Glycobiology 5: 505-10 (1991)). However, compositions that havedifferent structures in solution than the normal vesicular structure arealso encompassed. For example, the lipids may assume a micellarstructure or merely exist as non-uniform aggregates of lipid molecules.Also contemplated are lipofectamine-nucleic acid complexes.

Non-Viral Based Delivery Systems

In some instances, polypeptides described herein can also be introducedinto effector cells such as T cells using non-viral based deliverysystems, such as the “Sleeping Beauty (SB) Transposon System,” whichrefers a synthetic DNA transposon system to introduce DNA sequences intothe chromosomes of vertebrates. The system is described, for example, inU.S. Pat. Nos. 6,489,458 and 8,227,432.

The Sleeping Beauty transposon system is composed of a Sleeping Beauty(SB) transposase and a SB transposon. DNA transposons translocate fromone DNA site to another in a simple, cut-and-paste manner. Transpositionis a precise process in which a defined DNA segment is excised from oneDNA molecule and moved to another site in the same or different DNAmolecule or genome. As do other Tc1/mariner-type transposases, SBtransposase inserts a transposon into a TA dinucleotide base pair in arecipient DNA sequence. The insertion site can be elsewhere in the sameDNA molecule, or in another DNA molecule (or chromosome). In mammaliangenomes, including humans, there are approximately 200 million TA sites.The TA insertion site is duplicated in the process of transposonintegration. This duplication of the TA sequence is a hallmark oftransposition and used to ascertain the mechanism in some experiments.The transposase can be encoded either within the transposon or thetransposase can be supplied by another source, in which case thetransposon becomes a non-autonomous element. Non-autonomous transposonsare most useful as genetic tools because after insertion they cannotindependently continue to excise and re-insert. SB transposons envisagedto be used as non-viral vectors for introduction of genes into genomesof vertebrate animals and for gene therapy.

Briefly, the Sleeping Beauty (SB) system (Hackett et al., Mol Ther18:674-83, (2010)) was adapted to genetically modify the T cells (Cooperet al., Blood 105:1622-31, (2005)). This involved two steps: (i) theelectro-transfer of DNA plasmids expressing a SB transposon [i.e.,chimeric antigen receptor (CAR) to redirect T-cell specificity (Jin etal., Gene Ther 18:849-56, (2011); Kebriaei et al., Hum Gene Ther23:444-50, (2012)) and SB transposase and (ii) the propagation andexpansion of T cells stably expressing integrants on designer artificialantigen-presenting cells (AaPC) derived from the K562 cell line (alsoknown as AaPCs (Activating and Propagating Cells). In one embodiment,the SB transposon system includes coding sequence encoding mbIL-15, acell tag and/or a chimeric antigen receptor. In one embodiment, the SBtransposon system includes coding sequence encoding mbIL-15, a cell tagand/or a T-cell receptor (TCR). In another, embodiment, the second step(ii) is eliminated and the genetically modified T cells arecryopreserved or immediately infused into a patient. In certainembodiments, the genetically modified T cells are not cryopreservedbefore infusion into a patient.

Such systems are described for example in Hudecek et al., CriticalReviews in Biochemistry and Molecular Biology, 52:4, 355-380 (2017),Singh et al., Cancer Res (8):68 (2008). Apr. 15, 2008 and Maiti et al.,J Immunother. 36(2): 112-123 (2013), incorporated herein by reference intheir entireties.

In some embodiments, a modified effector cell (e.g., a CAR effector cellor a TCR effector cell) expressing a polypeptide described herein and acytokine such as IL-2, IL-22 or IL-15 for instance membrane-bound IL-15(mbIL-15) is encoded in a transposon DNA plasmid vector, and the SBtransposase is encoded in a separate vector. In specific embodiments, aCAR is encoded in a transposon DNA plasmid vector, mbIL-15 is encoded ina second transposon DNA plasmid vector, and the SB transposase isencoded in a third DNA plasmid vector. In some embodiments, the mbIL-15is encoded with a truncated epidermal growth factor receptor tag.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the polypeptides describedherein, in order to confirm the presence of the recombinant DNA sequencein the host cell, a variety of assays may be performed. Such assaysinclude, for example, molecular assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the instant disclosure.

In embodiments, a modified effector cell comprising a polynucleotideencoding a polypeptide described herein and other genetic elements aredelivered to a cell using the SB11 transposon system, the SB100Xtransposon system, the SB110 transposon system, the piggyBac transposonsystem (see, e.g., U.S. Pat. No. 9,228,180, Wilson et al, “PiggyBacTransposon-mediated Gene Transfer in Human Cells,” Molecular Therapy15:139-145 (2007), incorporated herein by reference in its entirety)and/or the piggyBat transposon system (see, e.g., Mitra et al.,“Functional characterization of piggyBat from the bat Myotis lucifugusunveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA110:234-239 (2013). Additional transposases and transposon systems areprovided in U.S. Pat. Nos. 7,148,203; 8,227,432; U.S. Patent Publn. No.2011/0117072; Mates et al., Nat Genet, 41(6):753-61 (2009). doi:10.1038/ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi:10.1038/gt.2011.40. Epub 2011 Mar. 31 and in Ivics et al., Cell.91(4):501-10, (1997), each of which is incorporated herein by referencein their entirety.

In other embodiments, a polypeptide described herein and other geneticelements such as cytokines, for example, mbIL-15 and/or a tag, can beintegrated into the immune effector cell's DNA through a recombinase andintegrating expression vectors. Such vectors can randomly integrate intothe host cell's DNA, or can include a recombination site to enable thespecific recombination between the expression vector and the host cell'schromosome. Such integrating expression vectors can utilize theendogenous expression control sequences of the host cell's chromosomesto effect expression of the desired protein. In some embodiments,targeted integration is promoted by the presence of sequences on thedonor polynucleotide that are homologous to sequences flanking theintegration site. For example, targeted integration using the donorpolynucleotides described herein can be achieved following conventionaltransfection techniques, e.g. techniques used to create gene knockoutsor knockins by homologous recombination. In other embodiments, targetedintegration is promoted both by the presence of sequences on the donorpolynucleotide that are homologous to sequences flanking the integrationsite, and by contacting the cells with donor polynucleotide in thepresence of a site-specific recombinase. By a site-specific recombinase,or simply a recombinase, it is meant a polypeptide that catalyzesconservative site-specific recombination between its compatiblerecombination sites. As used herein, a site-specific recombinaseincludes native polypeptides as well as derivatives, variants and/orfragments that retain activity, and native polynucleotides, derivatives,variants, and/or fragments that encode a recombinase that retainsactivity.

The recombinases can be introduced into a target cell before,concurrently with, or after the introduction of a targeting vector. Therecombinase can be directly introduced into a cell as a protein, forexample, using liposomes, coated particles, or microinjection.Alternately, a polynucleotide, either DNA or messenger RNA, encoding therecombinase can be introduced into the cell using a suitable expressionvector. The targeting vector components described above are useful inthe construction of expression cassettes containing sequences encoding arecombinase of interest. However, expression of the recombinase can beregulated in other ways, for example, by placing the expression of therecombinase under the control of a regulatable promoter (i.e., apromoter whose expression can be selectively induced or repressed).

A recombinase can be from the Integrase or Resolvase families. TheIntegrase family of recombinases has over one hundred members andincludes, for example, FLP, Cre, and lambda integrase. The Integrasefamily, also referred to as the tyrosine family or the lambda integrasefamily, uses the catalytic tyrosine's hydroxyl group for a nucleophilicattack on the phosphodiester bond of the DNA. Typically, members of thetyrosine family initially nick the DNA, which later forms a doublestrand break. Examples of tyrosine family integrases include Cre, FLP,SSV1, and lambda (k) integrase. In the resolvase family, also known asthe serine recombinase family, a conserved serine residue forms acovalent link to the DNA target site (Grindley, et al., (2006) Ann RevBiochem 16:16).

In one embodiment, the recombinase is an isolated polynucleotidesequence comprising a nucleic acid sequence that encodes a recombinaseselecting from the group consisting of a SPβc2 recombinase, a SF370.1recombinase, a Bxb1 recombinase, an A118 recombinase and ϕRv1recombinase. Examples of serine recombinases are described in detail inU.S. Pat. No. 9,034,652, hereby incorporated by reference in itsentirety.

Recombinases for use in the practice of the present invention can beproduced recombinantly or purified. Polypeptides having the desiredrecombinase activity can be purified to a desired degree of purity bymethods known in the art of protein ammonium sulfate precipitation,purification, including, but not limited to, size fractionation,affinity chromatography, HPLC, ion exchange chromatography, heparinagarose affinity chromatography (e.g., Thorpe & Smith, Proc. Nat. Acad.Sci. 95:5505-5510, 1998.)

In one embodiment, the recombinases can be introduced into theeukaryotic cells that contain the recombination attachment sites atwhich recombination is desired by any suitable method. Methods ofintroducing functional proteins, e.g., by microinjection or othermethods, into cells are well known in the art. Introduction of purifiedrecombinase protein ensures a transient presence of the protein and itsfunction, which is often a preferred embodiment. Alternatively, a geneencoding the recombinase can be included in an expression vector used totransform the cell, in which the recombinase-encoding polynucleotide isoperably linked to a promoter which mediates expression of thepolynucleotide in the eukaryotic cell. The recombinase polypeptide canalso be introduced into the eukaryotic cell by messenger RNA thatencodes the recombinase polypeptide. It is generally preferred that therecombinase be present for only such time as is necessary for insertionof the nucleic acid fragments into the genome being modified. Thus, thelack of permanence associated with most expression vectors is notexpected to be detrimental. One can introduce the recombinase gene intothe cell before, after, or simultaneously with, the introduction of theexogenous polynucleotide of interest. In one embodiment, the recombinasegene is present within the vector that carries the polynucleotide thatis to be inserted; the recombinase gene can even be included within thepolynucleotide.

In one embodiment, a method for site-specific recombination comprisesproviding a first recombination site and a second recombination site;contacting the first and second recombination sites with a prokaryoticrecombinase polypeptide, resulting in recombination between therecombination sites, wherein the recombinase polypeptide can mediaterecombination between the first and second recombination sites, thefirst recombination site is attP or attB, the second recombination siteis attB or attP, and the recombinase can be Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase or a Mycobacterium smegmatis phage recombinase, providedthat when the first recombination attachment site is attB, the secondrecombination attachment site is attP, and when the first recombinationattachment site is attP, the second recombination attachment site isattB

Further embodiments provide for the introduction of a site-specificrecombinase into a cell whose genome is to be modified. One embodimentrelates to a method for obtaining site-specific recombination in aneukaryotic cell comprises providing a eukaryotic cell that comprises afirst recombination attachment site and a second recombinationattachment site; contacting the first and second recombinationattachment sites with a prokaryotic recombinase polypeptide, resultingin recombination between the recombination attachment sites, wherein therecombinase polypeptide can mediate recombination between the first andsecond recombination attachment sites, the first recombinationattachment site is a phage genomic recombination attachment site (attP)or a bacterial genomic recombination attachment site (attB), the secondrecombination attachment site is attB or attP, and the recombinase isselected from the group consisting of a Listeria monocytogenes phagerecombinase, a Streptococcus pyogenes phage recombinase, a Bacillussubtilis phage recombinase, a Mycobacterium tuberculosis phagerecombinase and a Mycobacterium smegmatis phage recombinase, providedthat when the first recombination attachment site is attB, the secondrecombination attachment site is attP, and when the first recombinationattachment site is attP, the second recombination attachment site isattB. In an embodiment the recombinase is selected from the groupconsisting of an A118 recombinase, a SF370.1 recombinase, a SPβc2recombinase, ϕRv1 recombinase, and a Bxb1 recombinase. In one embodimentthe recombination results in integration.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell, in order to confirm the presence of the recombinant DNAsequence in the host cell, a variety of assays can be performed. Suchassays include, for example, “molecular biological” assays well known tothose of skill in the art, such as Southern and Northern blotting,RT-PCR and PCR; “biochemical” assays, such as detecting the presence orabsence of a particular peptide, e.g., by immunological means (ELISAsand Western blots) or by assays described herein to identify peptides orproteins or nucleic acids falling within the scope of the invention

Immune Effector Cell Sources

In certain aspects, the embodiments described herein include methods ofmaking and/or expanding antigen-specific redirected immune effectorcells (e.g., T-cells, NK-cell or NK T-cells) expressing polypeptidesdescribed herein, the methods comprising transfecting the cells with anexpression vector containing a DNA (or RNA) construct encoding thepolypeptide, then, optionally, stimulating the cells with feeder cells,recombinant antigen, or an antibody to the receptor to cause the cellsto proliferate. In certain aspects, the cell (or cell population)engineered to express a CAR or TCR is a stem cell, iPS cell, immuneeffector cell or a precursor of these cells.

Sources of immune effector cells can include both allogeneic andautologous sources. In some cases, immune effector cells may bedifferentiated from stem cells or induced pluripotent stem cells(iPSCs). Thus, cell for engineering according to the embodiments can beisolated from umbilical cord blood, peripheral blood, human embryonicstem cells, or iPSCs. For example, allogeneic T cells can be modified toinclude a chimeric antigen receptor (and optionally, to lack functionalTCR). In some aspects, the immune effector cells are primary human Tcells such as T cells derived from human peripheral blood mononuclearcells (PBMC). PBMCs can be collected from the peripheral blood or afterstimulation with G-CSF (Granulocyte colony stimulating factor) from thebone marrow, or umbilical cord blood. In some embodiments, G-CSFcomprises a peptide sequence at least 80% homology to the sequence shownin SEQ ID NO: 52 or a peptide sequence encoded by a nucleotide sequenceat least 80% homology to the sequence shown in SEQ ID NO: 146.

Following transfection or transduction (e.g., with a CAR expressionconstruct), the cells may be immediately infused or may becryo-preserved. In certain aspects, following transfection, the cellsmay be propagated for days, weeks, or months ex vivo as a bulkpopulation within about 1, 2, 3, 4, 5 days or more following genetransfer into cells. In a further aspect, following transfection, thetransfectants are cloned and a clone demonstrating presence of a singleintegrated or episomally maintained expression cassette or plasmid, andexpression of the chimeric antigen receptor is expanded ex vivo. Theclone selected for expansion demonstrates the capacity to specificallyrecognize and lyse antigen-expressing target cells.

The recombinant T cells may be expanded by stimulation with IL-2, orother cytokines that bind the common gamma-chain (e.g., IL-7, IL-12,IL-15, IL-21, and others). The recombinant T cells may be expanded bystimulation with artificial antigen presenting cells. The recombinant Tcells may be expanded on artificial antigen presenting cell or with anantibody, such as OKT3, which cross links CD3 on the T cell surface.Subsets of the recombinant T cells may be further selected with the useof magnetic bead based isolation methods and/or fluorescence activatedcell sorting technology and further cultured with the AaPCs. In afurther aspect, the genetically modified cells may be cryopreserved.

T cells can also be obtained from a number of sources, includingperipheral blood, bone marrow, lymph node tissue, cord blood, thymustissue, tissue from a site of infection, ascites, pleural effusion,spleen tissue, and tumor (tumor-infiltrating lymphocytes). In certainembodiments described herein, any number of T cell lines available inthe art, may be used. In certain embodiments, T cells can be obtainedfrom a unit of blood collected from a subject using any number oftechniques known to the skilled artisan, such as Ficoll® separation. Inembodiments, cells from the circulating blood of an individual areobtained by apheresis. The apheresis product typically containslymphocytes, including T cells, monocytes, granulocytes, B cells, othernucleated white blood cells, red blood cells, and platelets. In oneembodiment, the cells collected by apheresis may be washed to remove theplasma fraction and to place the cells in an appropriate buffer or mediafor subsequent processing steps. In one embodiment, the cells are washedwith phosphate buffered saline (PBS). In an alternative embodiment, thewash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations. Initial activation steps in the absence ofcalcium lead to magnified activation. As those of ordinary skill in theart would readily appreciate a washing step may be accomplished bymethods known to those in the art, such as by using a semi-automated“flow-through” centrifuge (for example, the Cobe 2991 cell processor,the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to themanufacturer's instructions. After washing, the cells may be resuspendedin a variety of biocompatible buffers, such as, for example, Ca²⁺-free,Mg²⁺-free PBS, PlasmaLyte A, or other saline solution with or withoutbuffer. Alternatively, the undesirable components of the apheresissample may be removed and the cells directly resuspended in culturemedia.

In another embodiment, T cells are isolated from peripheral bloodlymphocytes by lysing the red blood cells and depleting the monocytes,for example, by centrifugation through a PERCOLL® gradient or bycounterflow centrifugal elutriation. A specific subpopulation of Tcells, such as CD3⁺, CD28⁺, CD4⁺, CD8⁺, CD45RA⁺, and CD45RO⁺ T cells,can be further isolated by positive or negative selection techniques.For example, in one embodiment, T cells are isolated by incubation withanti-CD3/anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS®M-450 CD3/CD28 T, for a time period sufficient for positive selection ofthe desired T cells. In one embodiment, the time period is about 30minutes. In a further embodiment, the time period ranges from 30 minutesto 36 hours or longer and all integer values there between. In a furtherembodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. Inyet another embodiment, the time period is 10 to 24 hours. In oneembodiment, the incubation time period is 24 hours. For isolation of Tcells from patients with leukemia, use of longer incubation times, suchas 24 hours, can increase cell yield. Longer incubation times may beused to isolate T cells in any situation where there are few T cells ascompared to other cell types, such in isolating tumor infiltratinglymphocytes (TIL) from tumor tissue or from immune-compromisedindividuals. Further, use of longer incubation times can increase theefficiency of capture of CD8+ T cells. Thus, by simply shortening orlengthening the time T cells are allowed to bind to the CD3/CD28 beadsand/or by increasing or decreasing the ratio of beads to T cells (asdescribed further herein), subpopulations of T cells can bepreferentially selected for or against at culture initiation or at othertime points during the process. Additionally, by increasing ordecreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on thebeads or other surface, subpopulations of T cells can be preferentiallyselected for or against at culture initiation or at other desired timepoints. The skilled artisan would recognize that multiple rounds ofselection can also be used herein. In certain embodiments, it may bedesirable to perform the selection procedure and use the “unselected”cells in the activation and expansion process. “Unselected” cells canalso be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can beaccomplished with a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. One method is cellsorting and/or selection via negative magnetic immunoadherence or flowcytometry that uses a cocktail of monoclonal antibodies directed to cellsurface markers present on the cells negatively selected. For example,to enrich for CD4⁺ cells by negative selection, a monoclonal antibodycocktail typically includes antibodies to CD14, CD20, CD11b, CD16,HLA-DR, and CD8. In certain embodiments, it may be desirable to enrichfor or positively select for regulatory T cells which typically expressCD4⁺, CD25⁺, CD62L^(hi), GITR⁺, and FoxP3⁺. Alternatively, in certainembodiments, T regulatory cells are depleted by anti-CD25 conjugatedbeads or other similar method of selection.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion. Further, use of high cell concentrationsallows more efficient capture of cells that may weakly express targetantigens of interest, such as CD28-negative T cells, or from sampleswhere there are many tumor cells present (i.e., leukemic blood, tumortissue, etc.). Such populations of cells may have therapeutic value andwould be desirable to obtain. For example, using high concentration ofcells allows more efficient selection of CD8⁺ T cells that normally haveweaker CD28 expression.

In a related embodiment, it may be desirable to use lower concentrationsof cells. By significantly diluting the mixture of T cells and surface(e.g., particles such as beads), interactions between the particles andcells is minimized. This selects for cells that express high amounts ofdesired antigens to be bound to the particles. For example, CD4⁺ T cellsexpress higher levels of CD28 and are more efficiently captured thanCD8⁺ T cells in dilute concentrations. In one embodiment, theconcentration of cells used is 5×10⁶/ml. In other embodiments, theconcentration used can be from about 1×10⁵/ml to 1×10⁶/ml, and anyinteger value in between.

In other embodiments, the cells may be incubated on a rotator forvarying lengths of time at varying speeds at either 2-10° C. or at roomtemperature.

T cells for stimulation can also be frozen after a washing step. Afterthe washing step that removes plasma and platelets, the cells may besuspended in a freezing solution. While many freezing solutions andparameters are known in the art and will be useful in this context, onemethod involves using PBS containing 20% DMSO and 8% human serumalbumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20%Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25%Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human SerumAlbumin, and 7.5% DMSO or other suitable cell freezing media containingfor example, Hespan and PlasmaLyte A, the cells then are frozen to −80°C. at a rate of 1° per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

In certain embodiments, cryopreserved cells are thawed and washed asdescribed herein and allowed to rest for one hour at room temperatureprior to activation using the methods described herein.

Also contemplated is the collection of blood samples or apheresisproduct from a subject at a time period prior to when the expanded cellsas described herein might be needed. As such, the source of the cells tobe expanded can be collected at any time point necessary, and desiredcells, such as T cells, isolated and frozen for later use in T celltherapy for any number of diseases or conditions that would benefit fromT cell therapy, such as those described herein. In one embodiment ablood sample or an apheresis is taken from a generally healthy subject.In certain embodiments, a blood sample or an apheresis is taken from agenerally healthy subject who is at risk of developing a disease, butwho has not yet developed a disease, and the cells of interest areisolated and frozen for later use. In certain embodiments, the T cellsmay be expanded, frozen, and used at a later time. In certainembodiments, samples are collected from a patient shortly afterdiagnosis of a particular disease as described herein but prior to anytreatments. In a further embodiment, the cells are isolated from a bloodsample or an apheresis from a subject prior to any number of relevanttreatment modalities, including but not limited to treatment with agentssuch as natalizumab, efalizumab, antiviral agents, chemotherapy,radiation, immunosuppressive agents, such as cyclosporin, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or otherimmunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan,fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,FR901228, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin) (Liu et al., Cell 66:807-815, (1991); Henderson et al.,Immun 73:316-321, (1991); Bierer et al., Curr. Opin. Immun 5:763-773,(1993)). In a further embodiment, the cells are isolated for a patientand frozen for later use in conjunction with (e.g., before,simultaneously or following) bone marrow or stem cell transplantation, Tcell ablative therapy using either chemotherapy agents such as,fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, orantibodies such as OKT3 or CAMPATH. In another embodiment, the cells areisolated prior to and can be frozen for later use for treatmentfollowing B-cell ablative therapy such as agents that react with CD20,e.g., Rituxan.

In a further embodiment, T cells are obtained from a patient directlyfollowing treatment. In this regard, it has been observed that followingcertain cancer treatments, in particular treatments with drugs thatdamage the immune system, shortly after treatment during the period whenpatients would normally be recovering from the treatment, the quality ofT cells obtained may be optimal or improved for their ability to expandex vivo. Likewise, following ex vivo manipulation using the methodsdescribed herein, these cells may be in a preferred state for enhancedengraftment and in vivo expansion. Thus, it is contemplated to collectblood cells, including T cells, dendritic cells, or other cells of thehematopoietic lineage, during this recovery phase. Further, in certainembodiments, mobilization (for example, mobilization with GM-CSF) andconditioning regimens can be used to create a condition in a subjectwherein repopulation, recirculation, regeneration, and/or expansion ofparticular cell types is favored, especially during a defined window oftime following therapy. Illustrative cell types include T cells, Bcells, dendritic cells, and other cells of the immune system.

Activation and Expansion of Effector Cells

Whether prior to or after genetic modification of effector cells, forinstance T cells to express a desirable polypeptide described herein,the cells can be activated and expanded generally using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Patent Application Publication No.20060121005.

Generally, the effector cells described herein are expanded by contactwith a surface having attached thereto an agent that stimulates aCD3/TCR complex associated signal and a ligand that stimulates aco-stimulatory molecule on the surface of the cells. In particular,effector cell populations may be stimulated as described herein, such asby contact with an anti-CD3 antibody, or antigen-binding fragmentthereof, or an anti-CD2 antibody immobilized on a surface, or by contactwith a protein kinase C activator (e.g., bryostatin) in conjunction witha calcium ionophore. For co-stimulation of an accessory molecule on thesurface of the cells, a ligand that binds the accessory molecule isused. For example, a population of effector cells, for instance T cellscan be contacted with an anti-CD3 antibody and an anti-CD28 antibody,under conditions appropriate for stimulating proliferation of the Tcells. To stimulate proliferation of either CD4⁺ T cells or CD8⁺ Tcells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,France) can be used as can other methods commonly known in the art (Berget al., Transplant Proc. 30(8):3975-3977, (1998); Haanen et al., J. Exp.Med. 190(9):13191328, (1999); Garland et al., J. Immunol Meth.227(1-2):53-63, (1999)).

In certain embodiments, the primary stimulatory signal and theco-stimulatory signal for the effector cell may be provided by differentprotocols. For example, the agents providing each signal may be insolution or coupled to a surface. When coupled to a surface, the agentsmay be coupled to the same surface (i.e., in “cis” formation) or toseparate surfaces (i.e., in “trans” formation). Alternatively, one agentmay be coupled to a surface and the other agent in solution. In oneembodiment, the agent providing the co-stimulatory signal is bound to acell surface and the agent providing the primary activation signal is insolution or coupled to a surface. In certain embodiments, both agentscan be in solution. In another embodiment, the agents may be in solubleform, and then cross-linked to a surface, such as a cell expressing Fcreceptors or an antibody or other binding agent which will bind to theagents. In this regard, see for example, U.S. Patent ApplicationPublication Nos. 20040101519 and 20060034810 for artificial antigenpresenting cells (aAPCs) that are contemplated for use in activating andexpanding T cells.

In one embodiment, the two agents are immobilized on beads, either onthe same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By wayof example, the agent providing the primary activation signal is ananti-CD3 antibody or an antigen-binding fragment thereof and the agentproviding the co-stimulatory signal is an anti-CD28 antibody orantigen-binding fragment thereof; and both agents are co-immobilized tothe same bead in equivalent molecular amounts. In one embodiment, a 1:1ratio of each antibody bound to the beads for CD4+ T cell expansion andT cell growth is used. In certain aspects described herein, a ratio ofanti CD3:CD28 antibodies bound to the beads is used such that anincrease in T cell expansion is observed as compared to the expansionobserved using a ratio of 1:1. In one particular embodiment an increaseof from about 1 to about 3 fold is observed as compared to the expansionobserved using a ratio of 1:1. In one embodiment, the ratio of CD3:CD28antibody bound to the beads ranges from 100:1 to 1:100 and all integervalues there between. In one aspect described herein, more anti-CD28antibody is bound to the particles than anti-CD3 antibody, i.e., theratio of CD3:CD28 is less than one. In certain embodiments describedherein, the ratio of anti CD28 antibody to anti CD3 antibody bound tothe beads is greater than 2:1. In one particular embodiment, a 1:100CD3:CD28 ratio of antibody bound to beads is used. In anotherembodiment, a 1:75 CD3:CD28 ratio of antibody bound to beads is used. Ina further embodiment, a 1:50 CD3:CD28 ratio of antibody bound to beadsis used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody boundto beads is used. In embodiments, a 1:10 CD3:CD28 ratio of antibodybound to beads is used. In another embodiment, a 1:3 CD3:CD28 ratio ofantibody bound to the beads is used. In yet another embodiment, a 3:1CD3:CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1:500 to 500:1 and any integer valuesin between may be used to stimulate effector cells such as T cells orother target cells. As those of ordinary skill in the art can readilyappreciate, the ratio of particles to cells may depend on particle sizerelative to the target cell. For example, small sized beads could onlybind a few cells, while larger beads could bind many. In certainembodiments the ratio of cells to particles ranges from 1:100 to 100:1and any integer values in-between and in further embodiments the ratiocomprises 1:9 to 9:1 and any integer values in between, can also be usedto stimulate effector cells. The ratio of anti-CD3- andanti-CD28-coupled particles to T cells that result in T cell stimulationcan vary as noted above, however certain values include 1:100, 1:50,1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1 with one ratiobeing at least 1:1 particles per T cell. In one embodiment, a ratio ofparticles to cells of 1:1 or less is used. In one particular embodiment,the particle:cell ratio is 1:5. In further embodiments, the ratio ofparticles to cells can be varied depending on the day of stimulation.For example, in one embodiment, the ratio of particles to cells is from1:1 to 10:1 on the first day and additional particles are added to thecells every day or every other day thereafter for up to 10 days, atfinal ratios of from 1:1 to 1:10 (based on cell counts on the day ofaddition). In one particular embodiment, the ratio of particles to cellsis 1:1 on the first day of stimulation and adjusted to 1:5 on the thirdand fifth days of stimulation. In another embodiment, particles areadded on a daily or every other day basis to a final ratio of 1:1 on thefirst day, and 1:5 on the third and fifth days of stimulation. Inanother embodiment, the ratio of particles to cells is 2:1 on the firstday of stimulation and adjusted to 1:10 on the third and fifth days ofstimulation. In another embodiment, particles are added on a daily orevery other day basis to a final ratio of 1:1 on the first day, and 1:10on the third and fifth days of stimulation. One of skill in the art willappreciate that a variety of other ratios may be suitable for useherein. In particular, ratios will vary depending on particle size andon cell size and type.

In further embodiments described herein, the cells, such as T cells, arecombined with agent-coated beads, the beads and the cells aresubsequently separated, and then the cells are cultured. In analternative embodiment, prior to culture, the agent-coated beads andcells are not separated but are cultured together. In a furtherembodiment, the beads and cells are first concentrated by application ofa force, such as a magnetic force, resulting in increased ligation ofcell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowingparamagnetic beads to which anti-CD3 and anti-CD28 are attached (3×28beads) to contact the T cells. In one embodiment the cells (for example,10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 Tparamagnetic beads at a ratio of 1:1, or MACS® MicroBeads from MiltenyiBiotec) are combined in a buffer, for example, PBS (without divalentcations such as, calcium and magnesium). Again, those of ordinary skillin the art can readily appreciate any cell concentration may be used.For example, the target cell may be very rare in the sample and compriseonly 0.01% of the sample or the entire sample (i.e., 100%) may comprisethe target cell of interest. Accordingly, any cell number is within thecontext described herein. In certain embodiments, it may be desirable tosignificantly decrease the volume in which particles and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and particles. For example, in one embodiment, aconcentration of about 2 billion cells/ml is used. In anotherembodiment, greater than 100 million cells/ml is used. In a furtherembodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45,or 50 million cells/ml is used. In yet another embodiment, aconcentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mlis used. In further embodiments, concentrations of 125 or 150 millioncells/ml can be used. Using high concentrations can result in increasedcell yield, cell activation, and cell expansion. Further, use of highcell concentrations allows more efficient capture of cells that mayweakly express target antigens of interest, such as CD28-negative Tcells. Such populations of cells may have therapeutic value and would bedesirable to obtain in certain embodiments. For example, using highconcentration of cells allows more efficient selection of CD8+ T cellsthat normally have weaker CD28 expression.

In one embodiment described herein, the mixture may be cultured forseveral hours (about 3 hours) to about 14 days or any hourly integervalue in between. In another embodiment, the mixture may be cultured for21 days. In one embodiment described herein the beads and the T cellsare cultured together for about eight days. In another embodiment, thebeads and T cells are cultured together for 2-3 days. Several cycles ofstimulation may also be desired such that culture time of T cells can be60 days or more. Conditions appropriate for T cell culture include anappropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or,X-vivo 15, (Lonza)) that may contain factors necessary for proliferationand viability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-.gamma., IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGFbeta, and TNF-alpha or any other additives for thegrowth of cells known to the skilled artisan. Other additives for thegrowth of cells include, but are not limited to, surfactant, plasmanate,and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol.Media can include RPMI 1640, AIM-V, DMEM, MEM, alpha-MEM, F-12, X-Vivo15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate,and vitamins, either serum-free or supplemented with an appropriateamount of serum (or plasma) or a defined set of hormones, and/or anamount of cytokine(s) sufficient for the growth and expansion of Tcells. Antibiotics, e.g., penicillin and streptomycin, are included onlyin experimental cultures, not in cultures of cells that are to beinfused into a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

Effector cells, for instance T cells that have been exposed to variedstimulation times may exhibit different characteristics. For example,typical blood or apheresed peripheral blood mononuclear cell productshave a helper T cell population (T_(H), CD4⁺) that is greater than thecytotoxic or suppressor T cell population (T_(C), CD8⁺). Ex vivoexpansion of T cells by stimulating CD3 and CD28 receptors produces apopulation of T cells that prior to about days 8-9 consistspredominately of T_(H) cells, while after about days 8-9, the populationof T cells comprises an increasingly greater population of T_(C) cells.Accordingly, depending on the purpose of treatment, infusing a subjectwith a T cell population comprising predominately of T_(H) cells may beadvantageous. Similarly, if an antigen-specific subset of T_(C) cellshas been isolated it may be beneficial to expand this subset to agreater degree.

Further, in addition to CD4 and CD8 markers, other phenotypic markersvary significantly, but in large part, reproducibly during the course ofthe cell expansion process. Thus, such reproducibility enables theability to tailor an activated T cell product for specific purposes.

In some cases, immune effector cells of the embodiments (e.g., T-cells)are co-cultured with activating and propagating cells (AaPCs), to aid incell expansion. AaPCs can also be referred to as artificial AntigenPresenting cells (aAPCs). For example, antigen presenting cells (APCs)are useful in preparing therapeutic compositions and cell therapyproducts of the embodiments. In one aspect, the AaPCs may be transgenicK562 cells. For general guidance regarding the preparation and use ofantigen-presenting systems, see, e.g., U.S. Pat. Nos. 6,225,042,6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application PublicationNos. 2009/0017000 and 2009/0004142; and International Publication No.WO2007/103009, each of which is incorporated by reference. In yet afurther aspect of the embodiments, culturing the transgenic CAR cellscomprises culturing the transgenic CAR cells in the presence ofdendritic cells or activating and propagating cells (AaPCs) thatstimulate expansion of the CAR-expressing immune effector cells. Instill further aspects, the AaPCs comprise a CAR-binding antibody orfragment thereof expressed on the surface of the AaPCs. The AaPCs maycomprise additional molecules that activate or co-stimulate T-cells insome cases. The additional molecules may, in some cases, comprisemembrane-bound C7 cytokines. In yet still further aspects, the AaPCs areinactivated or irradiated, or have been tested for and confirmed to befree of infectious material. In still further aspects, culturing thetransgenic CAR cells in the presence of AaPCs comprises culturing thetransgenic CAR cells in a medium comprising soluble cytokines, such asIL-15, IL-21 and/or IL-2. The cells may be cultured at a ratio of about10:1 to about 1:10; about 3:1 to about 1:5; about 1:1 to about 1:3(immune effector cells to AaPCs); or any range derivable therein. Forexample, the co-culture of T cells and AaPCs can be at a ratio of about1:1, about 1:2 or about 1:3.

In one aspect, the AaPCs may express CD137L. In other aspects, the AaPCsmay further express CD19, CD64, CD86, or mIL15 (or mbIL-15). In certainaspects, the AaPCs may express at least one anti-CD3 antibody clone orits fragment, such as, for example, OKT3 and/or UCHT1. In one aspect,the AaPCs may be inactivated (e.g., irradiated or mitomycin C treated).In one aspect, the AaPCs may have been tested for and confirmed to befree of infectious material. Methods for producing such AaPCs are knownin the art. In one aspect, culturing the CAR-modified T cell populationwith AaPCs may comprise culturing the cells at a ratio of about 10:1 toabout 1:10; about 3:1 to about 1:5; about 1:1 to about 1:3 (T cells toAaPCs); or any range derivable therein. For example, the co-culture of Tcells and AaPCs can be at a ratio of about 1:1, about 1:2 or about 1:3.In one aspect, the culturing step may further comprise culturing with anaminobisphosphonate (e.g., zoledronic acid).

In a further aspect, the population of CAR expressing effector cells iscultured and/or stimulated for no more than 7, 14, 21, 28, 35 42 days,49, 56, 63 or 70 days. In some embodiments, the population of CAR-Tcells is cultured and/or stimulated for at least 0, 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30 or more days. In some embodiments,the population of CAR-T cells is cultured and/or stimulated for at least5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more days. In someembodiments, the population of CAR expressing effector cells is culturedand/or stimulated for at least 7, 14, 21, 28, 35, 42, 49, 56, 63 or moredays. In other embodiments, a stimulation includes the co-culture of theCAR expressing effector cells with AaPCs to promote the growth of CARpositive cells. In another aspect, the population of transgenic CARcells is stimulated for not more than: 1× stimulation, 2× stimulation,3× stimulation, 4× stimulation, 5× stimulation, 5× stimulation, 6×stimulation, 7× stimulation, 8× stimulation, 9× stimulation or 10×stimulation. In some instances, the transgenic cells are not cultured exvivo in the presence of AaPCs. In some specific instances, the method ofthe embodiment further comprises enriching the cell population forCAR-expressing immune effector cells (e.g., T-cells) after thetransfection and/or culturing step. The enriching may comprisefluorescence-activated cell sorting (FACS) and sorting forCAR-expressing cells. In a further aspect, the sorting forCAR-expressing cells comprises use of a CAR-binding antibody. Theenriching may also comprise depletion of CD56+ cells. In yet still afurther aspect of the embodiment, the method further comprisescryopreserving a sample of the population of transgenic CAR cells.

In some cases, AaPCs are incubated with a peptide of an optimal lengththat allows for direct binding of the peptide to the MHC moleculewithout additional processing. Alternatively, the cells can express anantigen of interest (i.e., in the case of MHC-independent antigenrecognition). Furthermore, in some cases, APCs can express an antibodythat binds to either a specific CAR polypeptide or to CAR polypeptidesin general (e.g., a universal activating and propagating cell (uAPC).Such methods are disclosed in WO/2014/190273, which is incorporatedherein by reference. In addition to peptide-MHC molecules or antigens ofinterest, the AaPC systems may also comprise at least one exogenousassisting molecule. Any suitable number and combination of assistingmolecules may be employed. The assisting molecule may be selected fromassisting molecules such as co-stimulatory molecules and adhesionmolecules. Exemplary co-stimulatory molecules include CD70 and B7.1(B7.1 was previously known as B7 and also known as CD80), which amongother things, bind to CD28 and/or CTLA-4 molecules on the surface of Tcells, thereby affecting, for example, T-cell expansion, Th1differentiation, short-term T-cell survival, and cytokine secretion suchas interleukin (IL)-2. Adhesion molecules may includecarbohydrate-binding glycoproteins such as selectins, transmembranebinding glycoproteins such as integrins, calcium-dependent proteins suchas cadherins, and single-pass transmembrane immunoglobulin (Ig)superfamily proteins, such as intercellular adhesion molecules (ICAMs),that promote, for example, cell-to-cell or cell-to-matrix contact.Exemplary adhesion molecules include LFA-3 and ICAMs, such as ICAM-1.Techniques, methods, and reagents useful for selection, cloning,preparation, and expression of exemplary assisting molecules, includingco-stimulatory molecules and adhesion molecules, are exemplified in,e.g., U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001, incorporatedherein by reference.

Cells selected to become AaPCs, preferably have deficiencies inintracellular antigen-processing, intracellular peptide trafficking,and/or intracellular MHC Class I or Class II molecule-peptide loading,or are poikilothermic (i.e., less sensitive to temperature challengethan mammalian cell lines), or possess both deficiencies andpoikilothermic properties. Preferably, cells selected to become AaPCsalso lack the ability to express at least one endogenous counterpart(e.g., endogenous MHC Class I or Class II molecule and/or endogenousassisting molecules as described above) to the exogenous MHC Class I orClass II molecule and assisting molecule components that are introducedinto the cells. Furthermore, AaPCs preferably retain the deficienciesand poikilothermic properties that were possessed by the cells prior totheir modification to generate the AaPCs. Exemplary AaPCs eitherconstitute or are derived from a transporter associated with antigenprocessing (TAP)-deficient cell line, such as an insect cell line. Anexemplary poikilothermic insect cells line is a Drosophila cell line,such as a Schneider 2 cell line (see, e.g., Schneider 1972 Illustrativemethods for the preparation, growth, and culture of Schneider 2 cells,are provided in U.S. Pat. Nos. 6,225,042, 6,355,479, and 6,362,001.

In one embodiment, AaPCs are also subjected to a freeze-thaw cycle. Inan exemplary freeze-thaw cycle, the AaPCs may be frozen by contacting asuitable receptacle containing the AaPCs with an appropriate amount ofliquid nitrogen, solid carbon dioxide (i.e., dry ice), or similarlow-temperature material, such that freezing occurs rapidly. The frozenAPCs are then thawed, either by removal of the AaPCs from thelow-temperature material and exposure to ambient room temperatureconditions, or by a facilitated thawing process in which a lukewarmwater bath or warm hand is employed to facilitate a shorter thawingtime. Additionally, AaPCs may be frozen and stored for an extendedperiod of time prior to thawing. Frozen AaPCs may also be thawed andthen lyophilized before further use. Preferably, preservatives thatmight detrimentally impact the freeze-thaw procedures, such as dimethylsulfoxide (DMSO), polyethylene glycols (PEGs), and other preservatives,are absent from media containing AaPCs that undergo the freeze-thawcycle, or are essentially removed, such as by transfer of AaPCs to mediathat is essentially devoid of such preservatives.

In further embodiments, xenogenic nucleic acid and nucleic acidendogenous to the AaPCs, may be inactivated by crosslinking, so thatessentially no cell growth, replication or expression of nucleic acidoccurs after the inactivation. In one embodiment, AaPCs are inactivatedat a point subsequent to the expression of exogenous MHC and assistingmolecules, presentation of such molecules on the surface of the AaPCs,and loading of presented MHC molecules with selected peptide orpeptides. Accordingly, such inactivated and selected peptide loadedAaPCs, while rendered essentially incapable of proliferating orreplicating, retain selected peptide presentation function. Preferably,the crosslinking also yields AaPCs that are essentially free ofcontaminating microorganisms, such as bacteria and viruses, withoutsubstantially decreasing the antigen-presenting cell function of theAaPCs. Thus crosslinking maintains the important AaPC functions of whilehelping to alleviate concerns about safety of a cell therapy productdeveloped using the AaPCs. For methods related to crosslinking andAaPCs, see for example, U.S. Patent Application Publication No.20090017000, which is incorporated herein by reference.

In certain embodiments there are further provided an engineered antigenpresenting cell (APC). Such cells may be used, for example, as describedabove, to propagate immune effector cells ex vivo. In further aspects,engineered APCs may, themselves be administered to a patient and therebystimulate expansion of immune effector cells in vivo. Engineered APCs ofthe embodiments may, themselves, be used as a therapeutic agent. Inother embodiments, the engineered APCs can used as a therapeutic agentthat can stimulate activation of endogenous immune effector cellsspecific for a target antigen and/or to increase the activity orpersistence of adoptively transferred immune effector cells specific toa target antigen.

As used herein the term “engineered APC” refers to cell(s) thatcomprises at least a first transgene, wherein the first transgeneencodes a HLA. Such engineered APCs may further comprise a secondtransgene for expression of an antigen, such that the antigen ispresented at the surface on the APC in complex with the HLA. In someaspects, the engineered APC can be a cell type that presented antigens(e.g., a dendritic cell). In further aspects, engineered APC can beproduced from a cell type that does not normally present antigens, sucha T-cell or T-cell progenitor (referred to as “T-APC”). Thus, in someaspects, an engineered APC of the embodiments comprises a firsttransgene encoding a target antigen and a second transgene encoding ahuman leukocyte antigen (HLA), such that the HLA is expressed on thesurface of the engineered APC in complex with an epitope of the targetantigen. In certain specific aspects, the HLA expressed in theengineered APC is HLA-A2.

In some aspects, an engineered APC of the embodiments may furthercomprise at least a third transgene encoding co-stimulatory molecule.The co-stimulatory molecule may be a co-stimulatory cytokine that may bea membrane-bound Cy cytokine. In certain aspects, the co-stimulatorycytokine is IL-15, such as membrane-bound IL-15. In some furtheraspects, an engineered APC may comprise an edited (or deleted) gene. Forexample, an inhibitory gene, such as PD-1, LIM-3, CTLA-4 or a TCR, canbe edited to reduce or eliminate expression of the gene. An engineeredAPC of the embodiments may further comprise a transgene encoding anytarget antigen of interest. For example, the target antigen can be aninfectious disease antigen or a tumor-associated antigen (TAA).

Point-of-Care

In one embodiment of the present disclosure, the immune effector cellsdescribed herein are modified at a point-of-care site. In one embodimentof the present disclosure, the immune effector cells described hereinare modified at or near a point-of-care site. In some cases, modifiedimmune effector cells are also referred to as engineered T cells. Insome cases, the point-of-care site is at a hospital or at a facility(e.g., a medical facility) near a subject in need of treatment. Thesubject undergoes apheresis and peripheral blood mononuclear cells(PBMCs) or a sub population of PBMC can be enriched for example, byelutriation, bead selection or Ficoll gradient separation. Enriched PBMCor a subpopulation of PBMC can be cryopreserved in any appropriatecryopreservation solution prior to further processing. In one instance,the elutriation process is performed using a buffer solution containinghuman serum albumin. Immune effector cells, such as T cells can beisolated by selection methods described herein. In one instance, theselection method for T cells includes beads specific for CD3 or beadsspecific for CD4 and CD8 on T cells. In one case, the beads can beparamagnetic beads. The harvested immune effector cells can becryopreserved in any appropriate cryopreservation solution prior tomodification. The immune effector cells can be thawed up to 24 hours, 36hours, 48 hours, 72 hours or 96 hours ahead of infusion. The thawedcells can be placed in cell culture, for example in cell culture (e.g.RPMI) supplemented with fetal bovine serum (FBS) or human serum AB orplaced in a buffer that includes cytokines such as IL-2 and IL-21, priorto modification. In another aspect, the harvested immune effector cellscan be modified without the need for cryopreservation.

In some cases, the immune effector cells are modified byengineering/introducing a chimeric receptor, one or more cell tag(s),and/or cytokine(s) into the immune effector cells and then rapidlyinfused into a subject. In some cases, the sources of immune effectorcells can include both allogeneic and autologous sources. In one case,the immune effector cells can be T cells or NK cells. In another case,the cytokine can be mbIL-15 or IL-12 or variants thereof. In furthercases, the cytokine can be modulated by ligand inducible gene-switchexpression systems described herein. For example, a ligand such asveledimex can be delivered to the subject to modulate the expression ofmbIL-15 or IL-12.

In another aspect, veledimex is provided at 5 mg, 10 mg, 15 mg, 20 mg,30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg or 100 mg. In a furtheraspect, lower doses of veledimex are provided, for example, 0.5 mg, 1mg, 5 mg, 10 mg, 15 mg or 20 mg. In one embodiment, veledimex isadministered to the subject 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 days prior to infusion of themodified immune effector cells. In a further embodiment, veledimex isadministered about once every 12 hours, about once every 24 hours, aboutonce every 36 hours or about once every 48 hours, for an effectiveperiod of time to a subject post infusion of the modified immuneeffector cells. In one embodiment, an effective period of time forveledimex administration is about: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days e.g., postimmune effector cell administration. In other embodiments, veledimex canbe re-administered after a rest period, after a drug holiday or when thesubject experiences a relapse.

In certain cases, where an adverse effect on a subject is observed orwhen treatment is not needed, the cell tag can be activated, for examplevia cetuximab, for conditional in vivo ablation of modified immuneeffector cells comprising cell tags such as truncated epidermal growthfactor receptor tags as described herein.

In some embodiments, such immune effectors cells are modified by theconstructs as described herein through electroporation. In one instance,electroporation is performed with electroporators such as Lonza'sNucleofector™ devices. In other embodiments, the vector comprising theabove-mentioned constructs is a non-viral or viral vector. In one case,the non-viral vector includes a Sleeping Beauty transposon-transposasesystem. In one instance, the immune effector cells are electroporatedusing a specific sequence. For example, the immune effector cells can beelectroporated with one transposon followed by the DNA encoding aSleeping Beauty transposase followed by a second transposon. In anotherinstance, the immune effector cells can be electroporated with alltransposons and transposase at the same time. In another instance, theimmune effector cells can be electroporated with a transposase followedby both transposons or one transposon at a time. While undergoingsequential electroporation, the immune effector cells can be rested fora period of time prior to the next electroporation step.

In some cases, the modified immune effector cells do not undergo apropagation and activation step. In some cases, the modified immuneeffector cells do not undergo an incubation or culturing step (e.g. exvivo propagation). In certain cases, the modified immune effector cellsare placed in a buffer that includes IL-2 and IL-21 prior to infusion.In other instances, the modified immune effector cells are placed orrested in cell culture buffer, for example in cell culture buffer (e.g.RPMI) supplemented with fetal bovine serum (FBS) prior to infusion.Prior to infusion, the modified immune effector cells can be harvested,washed and formulated in a saline buffer in preparation for infusioninto the subject.

In one instance, the subject has undergone lymphodepletion prior toinfusion. Exemplary lymphodepletion regimens can include theadministration of a fludarabine or cyclophosphamide or combinationthereof.

In other instances, lymphodepletion is not required and the modifiedimmune effector cells are rapidly infused into the subject.

In a further instance, the subject undergoes minimal lymphodepletion.Minimal lymphodepletion herein refers to a reduced lymphodepletionprotocol such that the subject can be infused within 1 day, 2 days or 3days following the lymphodepletion regimen. In one instance, a reducedlymphodepletion protocol can include lower doses of fludarabine and/orcyclophosphamide. In another instance, a reduced lymphodepletionprotocol can include a shortened period of lymphodepletion, for example1 day or 2 days.

In one embodiment, the immune effector cells are modified byengineering/introducing a chimeric receptor and a cytokine into saidimmune effector cells and then rapidly infused into a subject. In othercases, the immune effector cells are modified by engineering/introducinga chimeric receptor and a cytokine into said cells and then infusedwithin at least: 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 hours into a subject. In other cases, immune effector cells aremodified by engineering/introducing a chimeric receptor and a cytokineinto the immune effector cells and then infused in 0 days, <1 day, <2days, <3 days, <4 days, <5 days, <6 days or <7 days into a subject.

In some embodiments, an amount of modified effector cells isadministered to a subject in need thereof and the amount is determinedbased on the efficacy and the potential of inducing acytokine-associated toxicity. In another embodiment, the modifiedeffector cells are CAR⁺ and CD3+ cells. In some cases, an amount ofmodified effector cells comprises about 10⁴ to about 10⁹ modifiedeffector cells/kg. In some cases, an amount of modified effector cellscomprises about 10⁴ to about 10⁵ modified effector cells/kg. In somecases, an amount of modified effector cells comprises about 10⁵ to about10⁶ modified effector cells/kg. In some cases, an amount of modifiedeffector cells comprises about 10⁶ to about 10⁷ modified effectorcells/kg. In some cases, an amount of modified effector cells comprises>10⁴ but ≤10⁵ modified effector cells/kg. In some cases, an amount ofmodified effector cells comprises >10⁵ but ≤10⁶ modified effectorcells/kg. In some cases, an amount of modified effector cells comprises>10⁶ but ≤10⁷ modified effector cells/kg.

In one embodiment, the modified immune effector cells are targeted tothe cancer via regional delivery directly to the tumor tissue. Forexample, in ovarian cancer, the modified immune effector cells can bedelivered intraperitoneally (IP) to the abdomen or peritoneal cavity.Such IP delivery can be performed via a port or pre-existing port placedfor delivery of chemotherapy drugs. Other methods of regional deliveryof modified immune effector cells can include catheter infusion intoresection cavity, ultrasound guided intratumoral injection, hepaticartery infusion or intrapleural delivery.

In one embodiment, a subject in need thereof, can begin therapy with afirst dose of modified immune effector cells delivered via IP followedby a second dose of modified immune effector cells delivered via IV. Ina further embodiment, the second dose of modified immune effector cellscan be followed by subsequent doses which can be delivered via IV or IP.In one embodiment, the duration between the first and second or furthersubsequent dose can be about: 0, 1, 2, 3,4, 5, 6,7, 8,9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days.In one embodiment, the duration between the first and second or furthersubsequent dose can be about: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, or 36 months. In another embodiment, the durationbetween the first and second or further subsequent dose can be about: 0,1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years.

In another embodiment, a catheter can be placed at the tumor ormetastasis site for further administration of 1, 2, 3, 4, 5, 6, 7, 8, 9,10 doses of modified immune effector cells. In some cases, doses ofmodified effector cells can comprise about 10² to about 10⁹ modifiedeffector cells/kg. In cases where toxicity is observed, doses ofmodified effector cells can comprise about 10² to about 10⁵ modifiedeffector cells/kg. In some cases, doses of modified effector cells canstart at about 10² modified effector cells/kg and subsequent doses canbe increased to about: 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or 10⁹ modified effectorcells/kg.

In other embodiments, a method of stimulating the proliferation and/orsurvival of engineered cells comprises obtaining a sample of cells froma subject, and transfecting cells of the sample of cells with one ormore polynucleotides that comprise one or more transposons. In oneembodiment, the transposon(s) encode a chimeric antigen receptor (CAR)as described herein, a cytokine, one or more cell tags, and atransposase effective to integrate said one or more polynucleotides intothe genome of said cells, to provide a population of engineered cells.In an embodiment, the transposon(s) encode a chimeric antigen receptor(CAR) as described herein, a cytokine, one or more cell tags, geneswitch polypeptides for ligand-inducible control of the cytokine and atransposase effective to integrate said one or more polynucleotides intothe genome of said cells, to provide a population of engineered cells.In an embodiment, the gene switch polypeptides comprise i) a first geneswitch polypeptide that comprises a DNA binding domain fused to a firstnuclear receptor ligand binding domain, and ii) a second gene switchpolypeptide that comprises a transactivation domain fused to a secondnuclear receptor ligand binding domain. In some embodiments, the firstgene switch polypeptide and the second gene switch polypeptide areconnected by a linker. In one instance, lymphodepletion is not requiredprior to administration of the engineered cells to a subject.

In one instance, a method of in vivo expansion or propagation ofengineered cells comprises obtaining a sample of cells from a subject,and transfecting cells of the sample of cells with one or morepolynucleotides that comprise one or more transposons comprising thechimeric polypeptides described herein. In one embodiment, thetransposon(s) encode a chimeric antigen receptor (CAR) as describedherein, a cytokine, one or more cell tags, and a transposase effectiveto integrate said one or more polynucleotides into the genome of saidcells, to provide a population of engineered cells. In an embodiment,the transposon(s) encode a chimeric antigen receptor (CAR) as describedherein, a cytokine, one or more cell tags, gene switch polypeptides forligand-inducible control of the cytokine and a transposase effective tointegrate said one or more polynucleotides into the genome of saidcells, to provide a population of engineered cells. In an embodiment,the gene switch polypeptides comprise i) a first gene switch polypeptidethat comprises a DNA binding domain fused to a first nuclear receptorligand binding domain, and ii) a second gene switch polypeptide thatcomprises a transactivation domain fused to a second nuclear receptorligand binding domain. In some embodiments, the first gene switchpolypeptide and the second gene switch polypeptide are connected by alinker. In one instance, lymphodepletion is not required prior toadministration of the engineered cells to a subject.

In another embodiment, a method of enhancing in vivo persistence ofengineered cells in a subject in need thereof comprises obtaining asample of cells from a subject, and transfecting cells of the sample ofcells with one or more polynucleotides that comprise one or moretransposons comprising the chimeric polypeptides described herein. Insome cases, one or more transposons encode a chimeric antigen receptor(CAR), a cytokine, one or more cell tags, and a transposase effective tointegrate the DNA into the genome of said cells, to provide a populationof engineered cells. In some cases, one or more transposons encode achimeric antigen receptor (CAR), a cytokine, one or more cell tags, geneswitch polypeptides for ligand-inducible control of the cytokine and atransposase effective to integrate the DNA into the genome of saidcells, to provide a population of engineered cells. In some cases, thegene switch polypeptides comprise i) a first gene switch polypeptidethat comprises a DNA binding domain fused to a first nuclear receptorligand binding domain, and ii) a second gene switch polypeptide thatcomprises a transactivation domain fused to a second nuclear receptorligand binding domain, wherein the first gene switch polypeptide and thesecond gene switch polypeptide are connected by a linker. In oneinstance, lymphodepletion is not required prior to administration of theengineered cells to a subject.

In another embodiment, a method of treating a subject with a tumorcomprises obtaining a sample of cells from a subject, transfecting cellsof the sample with one or more polynucleotides that comprise one or moretransposons comprising the chimeric polypeptides described herein, andadministering the population of engineered cells to the subject. In oneinstance, lymphodepletion is not required prior to administration of theengineered cells to a subject. In some cases, the one or moretransposons encode a chimeric antigen receptor (CAR), a cytokine, one ormore cell tags, and a transposase effective to integrate the DNA intothe genome of the cells. In some cases, the one or more transposonsencode a chimeric antigen receptor (CAR), a cytokine, one or more celltags, gene switch polypeptides for ligand-inducible control of thecytokine and a transposase effective to integrate the DNA into thegenome of the cells. In some cases, the gene switch polypeptidescomprise: i) a first gene switch polypeptide that comprises a DNAbinding domain fused to a first nuclear receptor ligand binding domain,and ii) a second gene switch polypeptide that comprises atransactivation domain fused to a second nuclear receptor ligand bindingdomain, wherein the first gene switch polypeptide and second gene switchpolypeptide are connected by a linker. In some cases, the cells aretransfected via electroporation. In some cases, the polynucleotidesencoding the gene switch polypeptides are modulated by a promoter. Insome cases, the promoter is a tissue-specific promoter or an EF1Apromoter or functional variant thereof. In some cases, thetissue-specific promoter comprises a T cell specific response element oran NFAT response element. In some cases, the cytokine comprises at leastone of IL-1, IL-2, IL-15, IL-12, IL-21, a fusion of IL-15, IL-15Ralphaor an IL-15 variant. In some cases, the cytokine is in secreted form. Insome cases, the cytokine is in membrane-bound form. In some cases, thecells are NK cells, NKT cells, T-cells or T-cell progenitor cells. Insome cases, the cells are administered to a subject (e.g. by infusingthe subject with the engineered cells). In some cases, the methodfurther comprises administering an effective amount of a ligand (e.g.veledimex) to induce expression of the cytokine. In some cases, the CARis capable of binding at least ROR1. In some cases, the transposase issalmonid-type Tc1-like transposase. In some cases, the transposase isSB11 or SB100x transposase. In other cases, the transposase is PiggyBac.In some cases, the cell tag comprises at least one of a HER1 truncatedvariant or a CD20 truncated variant.

Therapeutic Applications

In embodiments described herein, is an immune effector cell (e.g., Tcell) transduced with Sleeping Beauty transposon(s) and Sleeping Beautytransposase. For example, the Sleeping Beauty transposon or transposonscan include a CAR that combines an antigen recognition domain with aspacer of CD8 alpha hinge wherein the spacer region comprises a stalkregion designated as “s” and at least one stalk extension region,designated as “s′-n,” wherein n represents the number of units of s′ inthe space region, and wherein n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, an intracellular domain ofCD3-zeta, CD28, 4-1BB, or any combinations thereof and the intracellulardomain CD3-zeta, one or more cell tags, one or more cytokines andoptionally, components of the gene switch system as described herein.Therefore, in some instances, the transduced T cell can elicit aCAR-mediated T-cell response.

In embodiments described herein, the use of a CAR is provided toredirect the specificity of a primary T cell to a surface antigen. Thus,the present invention also provides a method for stimulating a Tcell-mediated immune response to a target cell population or tissue in amammal comprising the step of administering to the mammal a T cell thatexpresses a CAR, wherein the CAR comprises a binding moiety thatspecifically interacts with an antigen, a spacer, a zeta chain portioncomprising for example the intracellular domain of human CD3-zeta, and acostimulatory signaling region.

In one embodiment, the present disclosure includes a cellular therapywhere T cells are genetically modified to express the antigen-specificCARs of the invention and the CAR T cell is infused to a recipient inneed thereof. The infused cell is able to kill cells overexpressing anantigen in the recipient. Unlike antibody therapies, CAR T cells asdescribed herein are able to replicate in vivo resulting in long-termpersistence that can lead to sustained effect on tumor cells.

The invention additionally provides a method for detecting a diseasethat comprises overexpression of an antigen in a subject, comprising a)providing i) a sample from a subject, and ii) any one or more of theantibodies, or antigen-binding fragments thereof, that are describedherein, b) contacting the sample with the antibody under conditions forspecific binding of the antibody with its antigen, and c) detecting anincreased level of binding of the antibody to the sample compared to acontrol sample lacking the disease, thereby detecting the disease in thesubject. In one embodiment, the disease is cancer. In a preferredembodiment, the cancer is selected from the group of ovarian cancer andbreast cancer. While not intending to limit the method of detection, inone embodiment, detecting binding of the antibody to the sample isimmunohistochemical, enzyme-linked immunosorbent assay (ELISA),fluorescence-activated cell sorting (FACS), Western blot,immunoprecipitation, and/or radiographic imaging.

Also provided herein is a method for treating a disease that comprisesoverexpression of an antigen, comprising administering to a subjecthaving the disease a therapeutically effective amount of any one or moreof the polypeptides that are described herein.

The modified T cells described herein can also serve as a type ofvaccine for ex vivo immunization and/or in vivo therapy in a mammal. Inembodiments, the mammal is a human. With respect to ex vivoimmunization, at least one of the following occurs in vitro prior toadministering the immune effector cell into a mammal: i) expansion ofthe cells, ii) introducing a nucleic acid encoding a CAR to the cells,and/or iii) cryopreservation of the cells.

Ex vivo procedures are well known and are discussed more fully below.Briefly, cells are isolated from a mammal (for example, a human) andgenetically modified (i.e., transduced or transfected in vitro) with avector expressing a CAR disclosed herein. The CAR-modified cell can beadministered to a mammalian recipient to provide a therapeutic benefit.The mammalian recipient can be a human and the CAR-modified cell can beautologous with respect to the recipient. Alternatively, the cells canbe allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitorcells is described in U.S. Pat. No. 5,199,942, incorporated herein byreference, can be applied to the cells of the present invention. Othersuitable methods are known in the art, therefore the present inventionis not limited to any particular method of ex vivo expansion of thecells. Briefly, ex vivo culture and expansion of T cells comprises: (1)collecting CD34+ hematopoietic stem and progenitor cells from a mammalfrom peripheral blood harvest or bone marrow explants; and (2) expandingsuch cells ex vivo. In addition to the cellular growth factors describedin U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 andc-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivoimmunization, the present invention also provides compositions andmethods for in vivo immunization to elicit an immune response directedagainst an antigen in a patient.

Generally, the cells activated and expanded as described herein can beutilized in the treatment and prevention of diseases that arise inindividuals who are immunocompromised. In particular, the modified Tcells of the invention are used in the treatment of malignancies. Incertain embodiments, the cells of the invention are used in thetreatment of patients at risk for developing malignancies. Thus, themethods for the treatment or prevention of malignancies comprisingadministering to a subject in need thereof, a therapeutically effectiveamount of the modified T cells of the invention. In embodiments, thecells activated and expanded as described herein can be utilized in thetreatment of malignancies

Briefly, pharmaceutical compositions described herein can comprise atarget cell population as described herein, in combination with one ormore pharmaceutically or physiologically acceptable carriers, diluentsor excipients. Such compositions can comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. In embodiments, compositions of thepresent invention are formulated for intravenous administration.

Pharmaceutical compositions described herein can be administered in amanner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages can be determined byclinical trials.

When “an immunologically effective amount”, or “therapeutic amount” isindicated, the precise amount of the compositions described herein to beadministered can be determined by a physician with consideration ofindividual differences in age, weight, and condition of the patient(subject). It can generally be stated that a pharmaceutical compositioncomprising the T cells described herein can be administered at a dosageof 10⁴ to 10⁹ cells/kg body weight, 10⁵ to 10⁶ cells/kg body weight,including all integer values within those ranges. T cell compositionscan also be administered multiple times at these dosages. The cells canbe administered by using infusion techniques that are commonly known inimmunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, (1988)). The optimal dosage and treatment regime for aparticular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it can be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom, and reinfuse thepatient with these activated and expanded T cells. This process can becarried out multiple times every few weeks. In certain embodiments, Tcells can be activated from blood draws of from 10 cc to 400 cc. Incertain embodiments, T cells are activated from blood draws of 20 cc, 30cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not to be boundby theory, using this multiple blood draw/multiple reinfusion protocolcan serve to select out certain populations of T cells. In anotherembodiment, it can be desired to administer activated T cells of thesubject composition following lymphodepletion of the patient, either viaradiation or chemotherapy.

The administration of compositions described herein can be carried outin any convenient manner, including by aerosol inhalation, injection,ingestion, transfusion, implantation or transplantation. Thecompositions described herein can be administered to a patientsubcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, orintraperitoneally. In one embodiment, the T cell compositions of thepresent invention are administered to a patient by intradermal orsubcutaneous injection. In another embodiment, the CAR-T cellcompositions of the present invention are administered by i.v.injection. The compositions of T cells can be injected directly into alymph node, or site of primary tumor or metastasis.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Forexample, the dose of the above treatment can be in the range of 1×10⁴CAR+ cells/kg to 5×10⁶ CAR+ cells/kg. Exemplary doses can be 1×10² CAR+cells/kg, 1×10³ CAR+ cells/kg, 1×10⁴ CAR+ cells/kg, 1×10⁵ CAR+ cells/kg,3×10⁵ CAR+ cells/kg, 1×10⁶ CAR+ cells/kg, 5×10⁶ CAR+ cells/kg, 1×10⁷CAR+ cells/kg, 1×10⁸ CAR+ cells/kg or 1×10⁹ CAR+ cells/kg. Theappropriate dose can be adjusted accordingly for an adult or a pediatricpatient.

Alternatively, a typical amount of immune effector cells administered toa mammal (e.g., a human) can be, for example, in the range of onehundred, one thousand, ten thousand, one million to 100 billion cells;however, amounts below or above this exemplary range are within thescope of the invention. For example, the dose of inventive host cellscan be about 1 million to about 50 billion cells (e.g., about 5 millioncells, about 25 million cells, about 500 million cells, about 1 billioncells, about 5 billion cells, about 20 billion cells, about 30 billioncells, about 40 billion cells, or a range defined by any two of theforegoing values), about 10 million to about 100 billion cells (e.g.,about 20 million cells, about 30 million cells, about 40 million cells,about 60 million cells, about 70 million cells, about 80 million cells,about 90 million cells, about 10 billion cells, about 25 billion cells,about 50 billion cells, about 75 billion cells, about 90 billion cells,or a range defined by any two of the foregoing values), about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells, or a range defined by any two of the foregoing values).

Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment isrepeated until a desired suppression of disease symptoms occurs.However, other dosage regimens can be useful and are within the scope ofthe invention. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The composition comprising the immune effector cells expressing thedisclosed nucleic acid sequences, or a vector comprising the thosenucleic acid sequences, can be administered with one or more additionaltherapeutic agents, which can be co-administered to the mammal. By“co-administering” is meant administering one or more additionaltherapeutic agents and the composition comprising the inventive hostcells or the inventive vector sufficiently close in time to enhance theeffect of one or more additional therapeutic agents, or vice versa. Inthis regard, the composition comprising the immune effector cellsdescribed herein or a vector described herein can be administeredsimultaneously with one or more additional therapeutic agents, or first,and the one or more additional therapeutic agents can be administeredsecond, or vice versa. Alternatively, the composition comprising thedisclosed immune effector cells or the vectors described herein and theone or more additional therapeutic agents can be administeredsimultaneously.

An example of a therapeutic agents that can be included in orco-administered with the composition (or included in kits) comprisingthe inventive host cells and/or the inventive vectors are interleukins,cytokines, interferons, adjuvants and chemotherapeutic agents. Inembodiments, the additional therapeutic agents are IFN-alpha, IFN-beta,IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, andhGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5,TLR6, TLR7, TLR8, TLR9, and TLR10.

“Antifoaming agents” reduce foaming during processing which can resultin coagulation of aqueous dispersions, bubbles in the finished film, orgenerally impair processing. Exemplary anti-foaming agents includesilicon emulsions or sorbitan sesquoleate.

“Antioxidants” include, for example, butylated hydroxytoluene (BHT),sodium ascorbate, ascorbic acid, sodium metabisulfite and tocopherol. Incertain embodiments, antioxidants enhance chemical stability whererequired.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

“Binders” impart cohesive qualities and include, e.g., alginic acid andsalts thereof; cellulose derivatives such as carboxymethylcellulose,methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose,hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®),ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g.,Avicel®); microcrystalline dextrose; amylose; magnesium aluminumsilicate; polysaccharide acids; bentonites; gelatin;polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone;starch; pregelatinized starch; tragacanth, dextrin, a sugar, such assucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol,xylitol (e.g., Xylitab®), and lactose; a natural or synthetic gum suchas acacia, tragacanth, ghatti gum, mucilage of isapol husks,polyvinylpyrrolidone (e.g., Polyvidone® CL, Kollidon® CL, Polyplasdone®XL-10), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodiumalginate, and the like.

A “carrier” or “carrier materials” include any commonly used excipientsin pharmaceutics and should be selected on the basis of compatibilitywith compounds disclosed herein, such as, compounds of ibrutinib and ananticancer agent, and the release profile properties of the desireddosage form. Exemplary carrier materials include, e.g., binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. “Pharmaceutically compatible carrier materials” may include, butare not limited to, acacia, gelatin, colloidal silicon dioxide, calciumglycerophosphate, calcium lactate, maltodextrin, glycerine, magnesiumsilicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters,sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine,sodium chloride, tricalcium phosphate, dipotassium phosphate, celluloseand cellulose conjugates, sugars sodium stearoyl lactylate, carrageenan,monoglyceride, diglyceride, pregelatinized starch, and the like. See,e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed(Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E.,Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., PharmaceuticalDosage Forms, Marcel Decker, New York, N.Y., 1980; and PharmaceuticalDosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams& Wilkins 1999).

“Dispersing agents,” and/or “viscosity modulating agents” includematerials that control the diffusion and homogeneity of a drug throughliquid media or a granulation method or blend method. In someembodiments, these agents also facilitate the effectiveness of a coatingor eroding matrix. Exemplary diffusion facilitators/dispersing agentsinclude, e.g., hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG,polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and thecarbohydrate-based dispersing agents such as, for example, hydroxypropylcelluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers (e.g., PluronicsF68®, F88®, and F108®, which are block copolymers of ethylene oxide andpropylene oxide); and poloxamines (e.g., Tetronic 908®, also known asPoloxamine 908®, which is a tetrafunctional block copolymer derived fromsequential addition of propylene oxide and ethylene oxide toethylenediamine (BASF Corporation, Parsippany, N.J.)),polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetatecopolymer (S-630), polyethylene glycol, e.g., the polyethylene glycolcan have a molecular weight of about 300 to about 6000, or about 3350 toabout 4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents particularly useful in liposomal dispersions and self-emulsifyingdispersions are dimyristoyl phosphatidyl choline, natural phosphatidylcholine from eggs, natural phosphatidyl glycerol from eggs, cholesteroland isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can used in the present compositions.

The term “diluent” refers to chemical compounds that are used to dilutethe compound of interest prior to delivery. Diluents can also be used tostabilize compounds because they can provide a more stable environment.Salts dissolved in buffered solutions (which also can provide pH controlor maintenance) are utilized as diluents in the art, including, but notlimited to a phosphate buffered saline solution. In certain embodiments,diluents increase bulk of the composition to facilitate compression orcreate sufficient bulk for homogenous blend for capsule filling. Suchcompounds include e.g., lactose, starch, mannitol, sorbitol, dextrose,microcrystalline cellulose such as Avicel*; dibasic calcium phosphate,dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate;anhydrous lactose, spray-dried lactose; pregelatinized starch,compressible sugar, such as Di-Pac® (Amstar); mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; mannitol, sodiumchloride; inositol, bentonite, and the like.

“Filling agents” include compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

“Lubricants” and “glidants” are compounds that prevent, reduce orinhibit adhesion or friction of materials. Exemplary lubricants include,e.g., stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, ahydrocarbon such as mineral oil, or hydrogenated vegetable oil such ashydrogenated soybean oil (Sterotex®), higher fatty acids and theiralkali-metal and alkaline earth metal salts, such as aluminum, calcium,magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes,Stearowet®, boric acid, sodium benzoate, sodium acetate, sodiumchloride, leucine, a polyethylene glycol (e.g., PEG-4000) or amethoxypolyethylene glycol such as Carbowax™, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica such as Syloid™, Cab-O-Silo, a starchsuch as corn starch, silicone oil, a surfactant, and the like.

“Plasticizers” are compounds used to soften the microencapsulationmaterial or film coatings to make them less brittle. Suitableplasticizers include, e.g., polyethylene glycols such as PEG 300, PEG400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propyleneglycol, oleic acid, triethyl cellulose and triacetin. In someembodiments, plasticizers can also function as dispersing agents orwetting agents.

“Solubilizers” include compounds such as triacetin, triethylcitrate,ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate,vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone,N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropylmethylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropylalcohol, cholesterol, bile salts, polyethylene glycol 200-600,glycofurol, transcutol, propylene glycol, and dimethyl isosorbide andthe like.

“Stabilizers” include compounds such as any antioxidation agents,buffers, acids, preservatives and the like.

“Suspending agents” include compounds such as polyvinylpyrrolidone,e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17,polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, e.g.,the polyethylene glycol can have a molecular weight of about 300 toabout 6000, or about 3350 to about 4000, or about 7000 to about 5400,sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxymethylcellulose acetate stearate,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

“Surfactants” include compounds such as sodium lauryl sulfate, sodiumdocusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitanmonooleate, polyoxyethylene sorbitan monooleate, polysorbates,polaxomers, bile salts, glyceryl monostearate, copolymers of ethyleneoxide and propylene oxide, e.g., Pluronic® (BASF), and the like. Someother surfactants include polyoxyethylene fatty acid glycerides andvegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; andpolyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10,octoxynol 40. In some embodiments, surfactants may be included toenhance physical stability or for other purposes.

“Viscosity enhancing agents” include, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof.

“Wetting agents” include compounds such as oleic acid, glycerylmonostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamineoleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitanmonolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate,sodium doccusate, triacetin, Tween 80, vitamin E TPGS, ammonium saltsand the like.

Kits/Article of Manufacture

Disclosed herein, in certain embodiments, are kits and articles ofmanufacture for use with one or more methods described herein. Such kitsinclude a carrier, package, or container that is compartmentalized toreceive one or more containers such as vials, tubes, and the like, eachof the container(s) comprising one of the separate elements to be usedin a method described herein. Suitable containers include, for example,bottles, vials, syringes, and test tubes. In one embodiment, thecontainers are formed from a variety of materials such as glass orplastic.

The articles of manufacture provided herein contain packaging materials.Examples of pharmaceutical packaging materials include, but are notlimited to, blister packs, bottles, tubes, bags, containers, bottles,and any packaging material suitable for a selected formulation andintended mode of administration and treatment.

For example, the container(s) include CAR-T cells (e.g., CD19 CAR-Tcells) described herein, and optionally in addition with cytokinesand/or chemotherapeutic agents disclosed herein. Such kits optionallyinclude an identifying description or label or instructions relating toits use in the methods described herein.

A kit typically includes labels listing contents and/or instructions foruse, and package inserts with instructions for use. A set ofinstructions will also typically be included.

In some embodiments, a label is on or associated with the container. Inone embodiment, a label is on a container when letters, numbers or othercharacters forming the label are attached, molded or etched into thecontainer itself; a label is associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. In one embodiment, a label is used toindicate that the contents are to be used for a specific therapeuticapplication. The label also indicates directions for use of thecontents, such as in the methods described herein.

Sequences

Provided below is a representative list of certain sequences included inembodiments provided herein.

Amino Acid Nucleotide Sequences Sequences SEQ ID NO SEQ ID NO CD8α 107 1(Homo sapiens) CD8α (C164S, C181S) 108 2 hinge CD8α hinge (CD8α 1X 109 3spacer) CD8α 2X spacer 110 4 CD8α 3X spacer 111 5 CD8α 3X V2 spacer 1126 CD8α 4X spacer 113 7 Whitlow Linker 114 8 GSG linker 115 9 SGSG linker116 10 (G4S)3 Linker 117 11 (G4S)3.CD8α Hinge spacer 118 12 Whitlowlinker.CD8α 119 13 Hinge spacer Whitlow linker(2x).CD8α 120 14 Hingespacer Whitlow linker.CD8α 121 15 Hinge(2x) spacer LNGFR extracellular122 16 domain (LNGFR 1X spacer) LNGFR 2X spacer 123 17 LNGFR Cys 2,3,4spacer 124 18 LNGFR Cys 2,3,4 2X 125 19 spacer LNGFR Cys3,4 spacer 12620 LNGFR Cys3,4 2X spacer 127 21 LNGFR Cys3,4 3X spacer 128 22 LNGFRCys3,4 4X spacer 129 23 CD8α transmembrane 130 24 domain CytotoxicT-lymphocyte 131 25 protein 4 transmembrane domain CD28 co-stimulatory132 26 endodomain 4-1BB (CD137) co- 133 27 stimulatory endodomain CD3zeta stimulatory 134 28 endodomain DNAX-activation protein 135 29 10(DAP 10) Signaling Domain DNAX-activation protein 136 30 12 (DAP12)Signaling Domain Homo sapiens CD28 137 31 CD28 hinge (CD28 1X 138 32spacer) CD28 2X spacer 139 33 CD28 3X spacer 140 34 CD28 4X spacer 14135 Homo sapiens Cytotoxic T- 142 36 lymphocyte protein 4 (CTLA-4) CTLA-41X spacer 37 CTLA-4 2X spacer 38 CTLA-4 3X spacer 39 CTLA-4 4X spacer 40Homo sapiens IgG3 hinge 41 Homo sapiens IgG4 hinge 42 Homo sapiens IgG4hinge 144 43 (S108P) (IgG4 1X spacer) IgG4 2X spacer 44 IgG4 3X spacer45 IgG4 4X spacer 46 IgG4 5X spacer 47 IgG4 6X spacer 48 Homo sapiensIgG4 hinge 145 49 (S108P)-CH2—CH3 spacer Homo sapiens IgG4 hinge 50(S108P)-CH3 spacer Homo sapiens B- 51 lymphocyte antigen CD19Granulocyte macrophage 146 52 colony-stimulating factor receptor alphaSignal Peptide Anti-CD19 monoclonal 147 53 antibody clone FMC63 variablelight chain Anti-CD19 monoclonal 148 54 antibody clone FMC63 variableheavy chain Anti-CD19 clone FMC63 149 55 single chain fragment variable(scFv) with Whitlow linker CD19-specific chimeric 150 56 antigenreceptor with CD8- 1X spacer (CD19-CD8a- CD28-CD3z) CD19-specificchimeric 151 57 antigen receptor with CD8- 2X spacer CD19-specificchimeric 152 58 antigen receptor with CD8- 3X spacer CD19-specificchimeric 153 59 antigen receptor with CD8- 3X v2 spacer CD19-specificchimeric 154 60 antigen receptor with CD8- 4X spacer Anti-CD33monoclonal 155 61 antibody clone hM195 variable light chain Anti-CD33monoclonal 156 62 antibody clone hM195 variable heavy chain Anti-CD33monoclonal 157 63 antibody clone hM195 scFv CD33-specific chimeric 15864 antigen receptor with CD8a hinge (CD8 1X) spacer CD33-specificchimeric 159 65 antigen receptor with CD8 2X spacer CD33-specificchimeric 160 66 antigen receptor with CD8 3X spacer CD33-specificchimeric 161 67 antigen receptor with CD8 3X V2 spacer CD33-specificchimeric 162 68 antigen receptor with CD8 4X spacer HUMAN T-cellreceptor 163 69 alpha (TCRα) chain constant (C) region Extracellularregion of 164 70 HUMAN T-cell receptor alpha (TCRα) chain constant (C)region TCRα TM domain 165 71 TCRα hinge (1X spacer) 166 72 TCRα (C96S)hinge 167 73 TCRα hinge.(G4S)3 Spacer 168 74 (G4S)3.TCRα hinge Spacer169 75 Whitlow linker.TCRα 170 76 hinge Spacer TCRα 2X spacer 171 77TCRα 3X spacer 172 78 TCRα 4X spacer 173 79 TCRα 2X V2 spacer 174 80TCRα 3X V2 spacer 175 81 TCRα 4X V2 spacer 176 82 HUMAN T-cell receptor177 83 beta-1 (TCRβ or TCRβ1) chain constant (C) region Extracellularregion of 178 84 HUMAN T-cell receptor beta-1 (TCRβ1) chain constant (C)region TCRβ TM domain 179 85 TCRβ hinge (1X spacer) 180 86 TCRβ (C131S)hinge 181 87 TCRβ hinge.(G4S)3 Spacer 182 88 (G4S)3.TCRβ hinge Spacer183 89 Whitlow linker.TCRβ hinge 184 90 Spacer TCRβ 2X spacer 185 91TCRβ 3X spacer 186 92 TCRβ 4X spacer 187 93 TCRβ 2X V2 spacer 188 94TCRβ 3X V2 spacer 189 95 TCRβ 4X V2 spacer 190 96 HUMAN T-cell receptor191 97 beta-2 (TCRβ2) chain constant (C) region Extracellular region of192 98 HUMAN T-cell receptor beta-2 (TCRβ2) chain constant (C) regionHUMAN T-cell receptor 193 99 gamma-1 (TCRγ1) chain constant (C) region 1Extracellular region of 194 100 HUMAN T-cell receptor gamma-1 (TCRγ1)chain constant (C) region 1 TCRγ1 transmembrane 195 101 domain HUMANT-cell receptor 196 102 gamma-2 (TCRγ2) chain constant (C) regionExtracellular region of 197 103 HUMAN T-cell receptor gamma-2 (TCRγ2)chain constant (C) region HUMAN T-cell receptor 198 104 delta (TCRδ)chain C region Extracellular region of 199 105 HUMAN T-cell receptordelta (TCRδ) chain C region TCRδ transmembrane 200 106 domain Anti-EGFRvIII Clone 139 201 202 VH Anti- EGFRvIII Clone 139 203 204 VL Anti-EGFRvIII Clone 205 206 MR1 VH Anti- EGFRvIII Clone 207 208 MR1 VL Anti-EGFRvIII Clone 209 210 MR1-1 VH Anti- EGFRvIII Clone 211 212 MR1-1 VLAnti- EGFRvIII Clone 213 214 humMR1-1 VH Anti- EGFRvIII Clone 215 216humMR1-1 VL Anti- EGFRvIII Clone 217 218 humMR1-2 VH Anti- EGFRvIIIClone 219 220 humMR1-2 VL Anti- EGFRvIII scFv Clone 221 222 139Anti-EGFRvIII scFv clone 223 224 MR1 Anti EGFRvIII scFv clone 225 226MR1-1 Anti-EGFRvIII scFv clone 227 228 huMR1-1 Anti-EGFRvIII scFv clone229 230 huMR1-2 EGFRVIII CAR (clone 139 231 232 scFv.CD8alphahinge&TM.4-1BB.CD3

 ) EGFRVIII CAR (MR1 233 234 scFv.CD8alpha hinge&TM.4-1BB.CD3

 ) EGFRVIII CAR (MR1-1 235 236 scFv.CD8alpha hinge&TM.4-1BB.CD3

 ) EGFRvIII CAR (humMR1- 237 238 1 scFv.CD8alpha hinge&TM.4-1BB.CD3

 ) EGFRvIII CAR (humMR1- 239 240 2 scFv.CD8alpha hinge&TM.4-1BB.CD3

 ) EGFRvIII CAR (MR1-1 241 242 scFv.CD8alpha 2x hinge&TM.4-1BB.CD3

 ) EGFRvIII CAR (MR1-1 243 244 scFv.CD8alpha 3x hinge&TM.4-1BB.CD3 

 ) EGFRVIII CAR (MR1-1 245 scFv.CD8alpha 4x 246 hinge&TM.4-1BB.CD3

 ) EGFRvIII CAR (huMR1-1 247 248 scFv.CD8alpha 3x hinge & TM.4-1BB.CD39

 ) EGFRvIII CAR (huMR1-1 249 250 scFv.CD8alpha 4x hinge & TM.4-1BB.CD3

 ) EGFRvIII CAR (huMR1-2 251 252 scFv.CD8alpha 3x hinge & TM.4-1BB.CD3

 ) EGFRvIII CAR (huMR1-2 253 254 scFv.CD8alpha 4x hinge & TM.4-1BB.CD3

EXAMPLES

These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein.

Example 1 Chimeric Antigen Receptors with CD8α-Derived Spacers ofVarying Lengths

Chimeric antigen receptors incorporating a spacer comprising a stalkregion and a stalk extension region(s) were generated. The stalk regioncomprised the sequence of the CD8α hinge region (SEQ ID NO: 3), and thestalk extension region(s) comprised 1, 2, or 3 regions. Each stalkextension region comprised an altered CD8α hinge region sequence (SEQ IDNO: 2) in which the disulfide-bond forming cysteine residues (boldresidues in Table 1) were converted to serines (underlined residues inTable 1). In the 2×, 3×, and 4× versions depicted in FIG. 2 , the stalkregion comprising CD8α hinge sequence retained the disulfide bondforming cysteines. This stalk region was downstream of the alteredregions lacking the cysteines. A second version of the 3× stalk (3× v2)was generated in which the stalk extension region retaining thedisulfide bond-forming cysteines was upstream of the stalk region andthe stalk extension region, both regions lacking cysteines. The aminoacid sequences of generated 1×, 2×, 3×, 4×, and 3× v2 stalks are listedin Table 1.

TABLE 1 CD8α spacer CD8α spacer Amino Acid Sequence terminologydescription SEQ ID NO: 1X Stalk only 3 2X Stalk and 1 4 stalk extensionregions; s’-1 3X Stalk and two 5 stalk extension- regions; s’-2 4X Stalkand three 7 stalk extension regions; s’-3 3X v2 Stalk and two 6 stalkextension- regions; s’-2

Example 2. Nucleofection of PBMC with SB System to Generate CD19-CAR-TCells with CD8α-Derived Spacers of Varying Lengths

DNA plasmids to express CD19-specific CARs with varying spacer lengthswere cloned in SB transposon vectors. SB transposons were transfectedinto peripheral blood mononuclear cells (PBMC) via nucleofection toredirect T cell specificity.

On day 0, 20 million PBMCs were resuspended in 100 μL of Amaxa Human Tcell Nucleofector solution (Lonza, Basel, Switzerland) mixed with atotal of 15 μg of transposons and 5 μg of transposase (SB11) andelectroporated.

The following day (day 1) cells were counted, and CAR expression wasmeasured by flow cytometry. CAR-T Cells were stimulated with either7-irradiated (100 Gy) or mitomycin C treated AaPCs at a 1:1 ratio. TheAaPC cells used were K562-AaPC expressing CD19 antigen. Cultures weresupplemented with IL-21 (30 ng/ml) only for the first round ofstimulation and subsequently with recombinant human IL-2 (50 IU/ml) andIL-21 (30 ng/ml) (Pepro Tech) for remaining stimulations. CAR-T cellcultures were phenotyped at the end of each stimulation cycle, whichtypically lasted 7 days. The cultures were phenotyped for CAR expressionby multi-parameter flow cytometry utilizing either Protein L oranti-idiotype antibody that recognizes CD19-specific CAR. Cultures werealso closely monitored for the outgrowth of NK cells (defined asCD3^(neg)CD56+ population) and were removed from the CAR T cell cultureswhen the percentage exceeded 10% of total cell populations usingmagnetic beads for specific for CD56 (Stem Cell Technologies and/orMiltenyi Biotec), according to the manufacturer's instructions.

Example 3. Expression of CD19-Specific CARs with CD8α-Derived Spacers ofVarying Lengths

T cells expressing CARs with CD19-specific antigen binding region andCD8α-derived spacers listed in Table 1 were generated.

The expression level of the CD19-specific CARs with varying spacerlengths was determined after successive rounds of stimulation byco-culture with AaPC as described in Example 2. CAR expression level wasdetermined by flow cytometry. For flow cytometry experiments, cells weregently resuspended and cell number and viability were measured usingTrypan blue exclusion method with the Countess instrument. 5×10⁵ cellsfor each of the samples were harvested at 330×g for 4 min. Harvestedcells were incubated on ice for at least 15 min with 10% human AB serumin HBSS. Antibody cocktails containing fluorescently conjugatedantibodies included one or more of antibodies specific to CD4 (CloneRPA-T4), CD8 (Clone SK1), CD3 (Clone UCHT1), CD56, and CD19-specific CAR(anti-idiotype antibody), in HBSS+0.1% BSA+2 mM EDTA. The preparedantibody cocktails and associated fluorescence minus one/isotype controlwere added to stain the cell samples, and then the samples wereincubated on ice for 30 min. The samples were then washed then with FACSbuffer (HBSS+0.5% BSA+2 mM EDTA) and stained with Fixable Viability Dye(eBiosciences) for 30 min on ice. Cells were washed with FACS buffer andthen fixed with a 4% paraformaldehyde solution (BD Cytofix; BDBiosciences). All samples were run on a LSR II flow cytometer, aFortessa X-20 flow cytometer (BD Biosciences) or iQue Screener Plus(Intellicyt) and data was analyzed using FlowJo V10 (TreeStar, Ashland,OR) or iQue Screener software. The data from two example experiments issummarized in Table 2. T-cell counts from ex vivo expansion were alsotracked and the corresponding data is depicted in FIG. 3 .

As shown in Table 2, improved CD19-specific CAR expression was observedas measured by % of T cells expressing CAR as well as Molecules ofEquivalent Soluble Fluorochrome (MESF) when spacers included stalkextension regions (CD8-2×, CD8-3× and CD8-4×) compared to when spaceronly included stalk region (CD8-1×). Similar level of expansion ofCD19-CAR-T cells with spacer of varying lengths was observed as shown inFIG. 3 . CD19-CAR-T cells with CD8-3× v2 spacer failed to express CAR onthe cell surface and failed to expand ex vivo upon co-culture with AaPC.Difference between CD8-3× and CD8-3× v2 spacer is the position of aminoacid substitutions to disrupt inter-chain disulfide bonds. This datasuggests that positioning of amino acid substitutions for dimerizationsites is critical for expression of proteins when using spacers ofvarying lengths.

Expression of CD19-specific CAR with (CD8-3×) or without (CD8-1×) stalkextension regions was compared in human T cells derived from PBMC ofanother healthy donor. CD19-specific CARs were introduced using SBsystem and CD19-CAR-T cells were propagated by co-culture with AaPC exvivo for 28 days as described in Example 2. T cells were analyzed byflow cytometry on day 1, 14, 21 and 28 post nucleofection to measureexpression of CD19-specific CAR. FIG. 3B captures % of T cells inculture that were positive for CD19-specific CAR and FIG. 3B capturesraw mean fluorescence intensity (MFI) of CD19-specific CAR. Datapresented in FIG. 3B shows that similar percentage of T cells showedCD19-specific CAR expression through the ex vivo expansion phase ofCAR-T cells for this donor. However, MFI was significantly higher whenCD19-specific CAR with stalk extension region (CD8-3×) compared towithout (CD8-1×) stalk extension region meaning improved expressionlevels of CAR when stalk extension region was utilized.

Expression of CD19-specific CAR with spacers of varying lengths was alsoconfirmed via Western Blot analysis (FIG. 4 ).

PBMC were nucleofected with plasmids of Sleeping Beauty system toexpress CD19-specific CAR of varying spacer lengths. Nucleofected cellswere cultured in presence of AaPC as described in Example 2.

After four rounds of stimulations, cell lysates were prepared forwestern blot analysis. Approximately 10 μg lysate/lane of NuPAGE 10%Bis-Tris gel was loaded. Proteins were transferred from gel to apolyvinylidene fluoride (PVDF) membranes using the iBlot® (LifeTechnologies) semi-dry transfer apparatus. Membrane was blocked using a5% (w/v) powdered milk solution in a PBS+Tween-20 (PBST; 1×PBS+0.05%Tween-20) solution stained with goat anti-human CD3ζ primary antibodyand rabbit anti-goat IgG HRP (KPL Laboratories) secondary antibody.SuperSignal™ West Pico Chemiluminescent Substrate (Thermo FisherScientific) for enhanced chemiluminescence (ECL) detection was utilized.

Image of the western blot was captured on the FluorChem™ E Imager(ProteinSimple, San Jose, CA) system using the Digital Darkroom softwareand AlphaView® software (ProteinSimple). FIG. 4 shows image of westernblot. As shown by arrows in FIG. 4 , increasingly higher molecularweight bands were detected by anti-CD3ζ antibody corresponding toincreased spacer lengths from stalk extension regions. Native CD3ζ bandsat expected molecular weight were also detected.

TABLE 2 Day 1 Day 7 Day 14 Day 21 Day 28 CD19 CAR % CAR MESF % CAR MESF% CAR MESF % CAR MESF % CAR MESF CD8-1X 42.00 28845 39.90 34068 51.3020984 77.80 21118 86.00 33611 CD8-2X 6.63 14595 76.90 178206 90.10 6958393.90 69875 97.50 94415 CD8-3X 12.50 10846 60.60 145928 86.30 4423191.30 54830 97.20 87394 CD8-4X 6.98 7319 63.50 158301 87.00 31716 91.0045239 97.20 57106 CD8-3X V2 0.17 123 0.41 236 1.16 460

Example 4. Cytotoxicity of CD19-CAR-T Cells with CD8α-Derived Spacers ofVarying Lengths

Cytotoxicity of CD19-CAR-T cells with CD8α derived spacers of varyinglengths towards K562 cell line modified to express CD19 antigen(K562/CD19) was measured in a 2 hr Europium release assay. K562/CD19target cell line was labeled using the DELFIA BATDA reagent (DELFIAEuTDA Cytotoxicity assay; Perkin Elmer). CD19-CAR-T effector (E) cellswere co-cultured with labeled K562/CD19 target (T) cells at (E:T) ratiosof 10:1, 5:1 2:2 or 1:1. After 2 hr, supernatant from the co-cultureswere harvested and developed with addition of the DELFIA Europium assayand read on a time-resolved fluorescence instrument to measurecytotoxicity of target cells. The results from example experiments aredepicted in FIG. 5A.

As shown in FIG. 5A, CD19-CAR-T cells with varying lengths of spacersshowed dose dependent cytotoxicity of K562/CD19 target cells. CD19-CAR-Tcells with CD8α derived spacers with stalk extension region(s) (CD8-2×,CD8-3× and CD8-4×) showed similar or improved cytotoxicity of K562/CD19cells compared to CD19-CAR-T cells lacking extended stalk region(CD8-1×). Cytotoxicity exerted by CD19-CAR-T cells with longer CD8αderived spacers (CD8-2×, CD8-3× and CD8-4×) was improved especially atlower E:T ratios (2:1 and 1:1) suggesting increased potency of theseCAR-T cells compared to CAR-T cells lacking stalk extension region(CD8-1×).

Furthermore, specificity of CD19-CAR-T cells with spacers of varyinglengths was demonstrated by co-culture assay with K562/CD19 cell line aswell as parental K562 (CD19^(neg)) as well as CD19^(neg) EL4 cell line.All target cell lines were labeled using the DELFIA BATDA reagent(DELFIA EuTDA Cytotoxicity assay; Perkin Elmer). CD19-CAR-T effector (E)cells were co-cultured with CD19′ or CD19^(neg) labeled target (T) cellsat (E:T) ratio of 10:1. After 2 hr, supernatant from co-cultures wereharvested and developed with addition of the DELFIA Europium assay andread on a time-resolved fluorescence instrument to measure cytotoxicityof target cells. Results from assay are shown in FIG. 5B.

As shown in FIG. 5B, compared to CD19-CAR-T cells with CD8-1× spacer,similar (CD8-2× and CD8-3×) or somewhat lower (CD8-4×) cytotoxicity ofK562/CD19 cell line was observed by CD19-CAR-T cells with extendedspacers. However, CD19-CAR-T cells with CD8-1× spacer showednon-specific cytotoxicity of CD19^(neg) parental K562 cell line in thisassay. Whereas, CD19-CAR-T cells with stalk extension regions did notshow non-specific cytotoxicity of CD19^(neg) parental K562 cell line.This may explain slightly lower cytotoxicity of K562/CD19 cells observedwith CD19-CAR-T cells with extended spacers. Importantly, CD19-CAR-Tcells with extended spacers exhibited higher CD19 target specificity.

In summary, FIG. 5 shows that CD19-CAR-T cells with stalk extensionregions displayed superior functionality compared to CD19-CAR-T cellslacking stalk extension region as shown by improved cytotoxicity ofCD19⁺ target cell lines especially at lower E:T ratios as well asimproved specificity towards CD19 antigen.

Example 5. Specific Cytokine Production by CD19-CAR-T Cells in Responseto CD19 Antigen Expressing Cells

Cytokine production was measured after co-culture of the CD19 CAR-Tcells with varying CD8α-derived spacer lengths to CD19+ or CD19^(neg)tumor cells. Briefly, CD19-CAR-T cells with varying CD8α-derived spacerswere co-cultured overnight with K562/CD19 (CD19+) or K562 (CD19^(neg))or EL4 (CD19^(neg)) cell lines at an E:T ratio of 10:1. Culturesupernatants were harvested for multiplex cytokine analysis using theQBeads® (Intellicyt). The multiplex analysis was performed forexpression of human IFNγ, IL-2 and TNF present in harvested culturemedia. Data from an example experiment is depicted in FIGS. 6A-6C.

Significantly higher levels of IFNγ, IL-2 and TNF cytokines weredetected following co-cultures with K562/CD19 cell line whenCD19-specific CAR included CD8α-derived stalk extension region comparedto CD19-specific CAR that only included CD8α hinge stalk region.Elevated cytokine response of all tested CD19-CAR-T cells was inspecific response to CD19 antigen present on surface of K562/CD19 cellline as CD19^(neg) cell lines failed to induce a cytokine response.Values plotted represent mean±SD of samples tested in duplicates.

In summary, FIG. 6 shows that CD19-CAR-T cells with stalk extensionregions displayed superior cytokine secretion compared to CD19-CAR-Tcells lacking stalk extension region in response to CD19 expressingtumor cells.

Example 6. Characterization of CD33-CAR-T Cells with CD8α-DerivedSpacers of Varying Lengths

T cells expressing CARs with CD33-specific antigen binding region andCD8α-derived spacers listed in Table 1 were generated by SB system.

Briefly, CD33-specific CAR vectors were introduced into PBMC viaelectroporation, using a Sleeping Beauty-based transposon system tomediate genomic integration of the transposons. On day 0, 20 millionPBMC were suspended in 100 μL of Amaxa human T cell Nucleofectorsolution (Cat. no. VPA-1002; Lonza, Basel, Switzerland) mixed with 15 μgof CAR transposon and 5 μg of SB transposase and electroporated. Thefollowing day (day1) cells count and viability were measured followed byflow cytometry to quantify CAR expression. CAR-T cells were stimulatedwith either T-irradiated (100 Gy) or mitomycin C treated AaPCs at a 1:1ratio. The AaPC cells used were K562 cell line expressing CD33 antigen.CD33-CAR-T cells were expanded ex vivo by once weekly stimulation withthe AaPCs at a 1:1 ratio. Cultures were maintained in IL-2 (50 IU/ml)and/or IL-21 (30 ng/ml). CD33-specific CAR expression was measured usingrecombinant CD33/Fc protein staining as detected by multi-parameter flowcytometry.

The expression level of CD33-specific CARs with varying spacer lengthsfrom in T cells from two healthy donors is summarized in Table 3 andFIG. 9 .

As shown in Table 3, improved CD33-specific CAR expression was observedpost gene transfer as measured by % of T cells expressing CAR whenspacer included CD8α-derived stalk extension region (CD8-3×) compared towhen spacer only included CD8α stalk region (CD8-1×). As observed withCD19 CAR-T cells, CD33-CAR-T cells with CD8-3× v2 spacer failed to showsignificant CAR expression on T cell surface as well as failed to expandupon co-culture with AaPC. Difference between CD8-3× and CD8-3× v2spacer is the position of amino acid substitutions to disruptinter-chain disulfide bonds. This data suggests that positioning ofamino acid substitutions is critical for expression of proteins usingspacers of varying lengths.

TABLE 3 Donor # 1 Donor # 2 CD33 CAR Spacer Length % CD3⁺ CAR⁺ % CD3⁺CAR⁺ CD8-1X 50.4 74.3 CD8-2X 29.3 82.9 CD8-3X 63.5 80.0 CD8-4X 63.6 80.7CD8-3X V2 19.5 9.03

Example 7. Characterization of ROR-1 CAR T Cells with Spacers of VaryingLengths

As T cells expressing CARs with ROR-1-specific antigen binding regionand CD8α-derived spacers listed in Table 1, as well as LNGFR ECD spacer(Table 4) were generated using SB system using method explained inExample 2.

The expression level of ROR-1 CARs with varying stalk lengths wasdetermined using flow cytometry by staining with ROR-1-Fc fusion proteinusing method similar to explained in Example 6. Data from two differenthealthy donor cells is depicted in FIGS. 7A-B. As shown in FIGS. 7A and7B, ROR-1 CAR without stalk extension region (CD8-1×) failed to expresson surface of T cells. ROR-1 CAR without stalk extension region (CD8-1×)also failed to expand in ex vivo culture (data not shown). ROR-1 CARwith stalk extension regions (CD8-2×, CD8-3× and CD8-4×) showed highlevels of CAR expression on cell surface. In summary, ROR-1 CAR requiresstalk extension region(s) to allow for expression of CAR on T cellsurface.

T cells expressing CARs with ROR-1-specific antigen binding region weregenerated with CD8α-derived stalks listed in Table land CD28-CD3zetacostimulatory domain using SB system using method explained in Example2. These CAR-T cells were assessed for their functional activity againstROR-1 expressing tumor cells.

CD107a, also known as lysosomal-associate membrane protein-1 (LAMP-1),is constitutively expressed in the late endosomes-lysosomes of cells buttransiently expressed on the cell surface on degranulating cells. Thedegranulation assay was established to assess the capability of theROR-1 CAR-T cells with CD8-3× spacer to recognize target cells with orwithout ROR-1 expression with concurrent intracellular IFNγ detection ona per cell basis. ROR-1 CAR-T cells were co-cultured with target cellsat a 10:1 E:T ratio. Target cell included EL4 (ROR-1^(neg)), EL4-ROR-1(ROR-1⁺). At the start of the co-culture, the fluorescently conjugatedCD107a or isotype antibody was added along with the Transport InhibitorCocktail (containing monensin and brefeldin, 1×; eBioscience) andincubated at 37° C. for 4 hrs. At the end of the incubation period,cells were pelleted in the plate and cell surface antigens were stainedfor detection of CAR expression and T cell markers. Following cellsurface staining, cells were also stained with the Fixable Cellviability dye (eBioscience) according to the manufacturer's instructionsthen washed followed by fixation with Fix/Perm Solution (BDBiosciences). After fixing the samples, cells were washed in a Perm/Washsolution (BD Biosciences) then intracellularly stained with thefluorescently conjugated anti-human IFNγ antibody. Samples were washedthen resuspended in appropriate staining buffer with data acquired on aLSR II flow cytometer (BD Biosciences). Data from an example experimentis depicted in FIGS. 8A-8B. ROR-1 CAR-T cells with CD8-3× spacer showedantigen specific degranulation and IFNγ expression.

Example 8. Chimeric Antigen Receptors with LNGFR-Derived Spacers ofVarying Lengths

Chimeric antigen receptors comprising a stalk region and a stalkextension region were generated. The spacer region comprised thesequence of the LNGFR ECD (SEQ ID NO: 17), LNGFR Cys 2, 3, 4 (SEQ ID NO:18) or Cys 3, 4 (SEQ ID NO: 19) as shown in Table 4.

TABLE 4 Amino Acid Sequence LNGFR spacer SEQ ID NO: ECD 16 Cys 2, 3, 418 Cys 3, 4 20

Example 9. Chimeric Antigen Receptors with CD28-Derived Spacers ofVarying Lengths

Chimeric antigen receptors comprising a stalk region and a stalkextension region were generated. The stalk region comprised the sequenceof the CD28 hinge region (SEQ ID NO: 32), and the stalk extension region(s′-n), wherein n=1, 2, or 3, wherein each stalk extension region of analtered CD28 hinge region in which the disulfide-bond forming cysteineresidues (bold residues in Table 5) were converted to serines(underlined residues in Table 5). In the 2× (SEQ ID NO: 33), 3× (SEQ IDNO: 34), and 4× (SEQ ID NO: 35) versions, the CD28 hinge regionretaining the disulfide bond forming cysteines was downstream of thealtered regions lacking the cysteines. The generated CD28-derived 1×,2×, 3×, and 4× stalks comprising (s′-n), wherein n=0, 1, 2, and 3respectively are listed in Table 5.

TABLE 5 CD28 Amino Acid Sequence spacer SEQ ID NO: 1X 32 2X 33 3X 34 4X35

Example 10. Chimeric Antigen Receptors with CTLA-4-Derived Spacers ofVarying Lengths

Chimeric antigen receptors comprising a stalk region and a stalkextension region were generated. The stalk region comprised the sequenceof the CTLA-4 hinge region (SEQ ID NO: 37), and the stalk extensionregion (s′-n), n=1, 2, or 3, and each stalk extension region an alteredCLTA-4 hinge region in which the disulfide-bond forming cysteineresidues (bold residues in Table 6) were converted to serines(underlined residues in Table 6). In the 2× (SEQ ID NO: 38), 3× (SEQ IDNO: 39), and 4× (SEQ ID NO: 40) versions, the CTLA-4 hinge regionretaining the disulfide bond forming cysteines was downstream of thealtered regions lacking the cysteines. The generated CTLA-4-derived 1×,2×, 3×, and 4× stalks comprising (s′-n), n=0, 1, 2, and 3 respectivelyare listed in Table 6.

TABLE 6 CTLA-4 Amino Acid Sequence spacer SEQ ID NO: 1X 37 2X 38 3X 394X 40

Example 10. T Cell Receptor (TCR) with TCRα and TCRβ Hinge DomainDerived Spacers of Varying Lengths

T cell receptor (TCR) α and β chains comprising a stalk region and astalk extension region were generated. The stalk region for TCRα chaincomprised the sequence of the TCRα hinge region (SEQ ID NO: 72), and thestalk extension region (s′-n), n=1, 2, or 3, and each stalk extensionregion of an altered TCRα hinge region in which the disulfide-bondforming cysteine residues (bold residues in Table 7) were converted toserine (underlined residues in Table 7). In the 2× (SEQ ID NO: 77), 3×(SEQ ID NO: 78), and 4× (SEQ ID NO: 79) versions shown in Table 7, theTCRα hinge region retaining the disulfide bond forming cysteine wasdownstream (C-terminal) of the altered regions lacking the cysteines.The generated TCRα hinge region derived 1×, 2×, 3×, and 4× stalkscomprising (s′-n), n=0, 1, 2, and 3, respectively, are listed in Table7.

TABLE 7 TCRα Amino Acid Sequence spacer SEQ ID NO: 1X 72 2X 77 3X 78 4X79

The stalk region for TCRβ chain comprised the sequence of the TCRβ hingeregion (SEQ ID NO: 86), and the stalk extension region (s′-n), n=1, 2,or 3, and each stalk extension region of an altered TCRβ hinge region inwhich the disulfide-bond forming cysteine residues (bold residues inTable 8) were converted to serine (underlined residues in Table 8) (SEQID NO: 87). In the 2× ((SEQ ID NO: 91), 3× (SEQ ID NO: 92), and 4× (SEQID NO: 93) versions shown in Table 8, the TCRβ hinge region retainingthe disulfide bond forming cysteine was downstream (C-terminal) of thealtered regions lacking the cysteines. The generated TCRβ hinge regionderived 1×, 2×, 3×, and 4× stalks comprising (s′-n), n=0, 1, 2, and 3respectively, are listed in Table 8.

TABLE 8 TCRβ Amino Acid Sequence spacer SEQ ID NO: 1X 86 2X 91 3X 92 4X93

Example 11. Expression of EGFRvIII Specific CARs with CD8α-DerivedSpacers of Varying Lengths

T cells expressing CARs with EGFRvIII-specific antigen binding region(MR1-1 and huMR1-1) and CD8α-derived spacers listed in Table 1 weregenerated by SB system.

Briefly, EGFRvIII-specific CAR vectors were introduced into viaelectroporation, using a Sleeping Beauty-based transposon system tomediate genomic integration of the transposons. On day 0, PBMC weremixed with CAR transposon and SB transposase and electroporated. Thefollowing day (day1) cells count and viability were measured followed byflow cytometry to quantify CAR expression. CAR-T cells were stimulatedby co-culture with either 7-irradiated) or mitomycin C treated AaPCs.The AaPC cells used were K562 cell line expressing EGFRvIII antigen.EGFRvIII-CAR-T cells were expanded ex vivo by once weekly stimulationwith the AaPCs. Cultures were maintained in media supplemented with IL-2(and/or IL-21. EGFRvIII-specific CAR expression was measured usingrecombinant EGFRvIII/Fc protein staining as detected by multi-parameterflow cytometry.

The expression level of EGFRvIII-specific CARs with varying spacerlengths from in T cells from two healthy donors is summarized in FIG.11A-B. One day after nucleofection, similar levels of EGFRvIII-specificCAR of different CD8α spacer lengths was observed using either MR1-1 orhuMR1-1 based CARs (FIG. 11A). However, only CAR-T cells expressingEGFRvIII-specific CARs with longer CD8α spacer lengths (3× and 4×) couldbe enriched via EGFRvIII antigen specific stimulation using AaPCco-culture (FIG. 11B) showing importance of longer spacers forinteraction with EGFRvIII antigen on surface of AaPC.

Example 12. Expansion of EGFRvIII Specific CARs with CD8α-DerivedSpacers of Varying Lengths

CAR-T cells undergo in vivo expansion after infusion upon recognition oftumor cells expressing antigen that CAR recognizes. In vivo expansion ofCAR-T cells is very important for their anti-tumor activity. Ex vivoexpansion via co-culture with antigen specific cell line, e.g. AaPC,simulates expansion of CAR-T cells in absence of tumor cells. Ex vivoexpansion of CAR-T cells is often performed during manufacturing toobtain sufficient CAR⁺ T cells for patient treatment.

EGFRvIII-specific CAR-T cells were expanded in vitro by recurringstimulations using co-culture with an AaPC cell line expressing EGFRvIIIantigen following gene transfer with SB derived transposon/transposaseas described in Example 11. Total numbers of EGFRvIII-specific CAR⁺ Tcells and their fold expansion in ex vivo culture following each AaPCstimulation were measured. As shown in FIG. 11C, CAR-T cells expressingEGFRvIII-specific CARs with longer CD8α spacer lengths (3× and 4×)showed robust expansion after four AaPC stimulations. However CAR-Tcells expressing EGFRvIII-specific CARs with 1× CD8α spacer failed toexpand. Robust expansion of CAR-T cells with longer CD8α spacer lengths(3× and 4×) is further evident with >200 fold expansion upon four AaPCstimulations (FIG. 11D).

Example 13. Cytotoxicity of EGFRvIII CAR T Cells with Spacers of VaryingLengths

Cytotoxicity of huMR1-1 EGFRvIII-specific CAR-T cells with CD8α derivedspacers of varying lengths towards a K562 cell line modified to expressEGFRvIII antigen (K562-EGFRvIII) was measured in a 2 hr Europium releaseassay. K562 parental cell line which does not express EGFRvIII was usedas control. K562 and K562-EGFRvIII target cell lines were labeled usingthe DELFIA BATDA reagent. EGFRvIII-specific CAR-T effector (E) cellswere co-cultured with labeled K562-EGFRvIII target (T) cells at (E:T)ratios of 10:1, 5:1 or 1:1. After 2 hr, supernatant from the co-cultureswere harvested and developed with addition of the DELFIA Europium assayand read on a time-resolved fluorescence instrument to measurecytotoxicity of target cells. The results from example experiments aredepicted in FIG. 11E.

As shown in FIG. 11E, EGFRvIII-specific CAR-T cells with varying lengthsof spacers showed dose dependent cytotoxicity of K562-EGFRvIII targetcells. Very low background cytotoxicity of K562 cells which do notexpress EGFRvIII was observed except for at highest E:T ratio of 10:1which may be due to high concentrations of effector cells present.EGFRvIII-specific CAR-T cells with CD8α derived spacers with stalkextension region(s) (CD8-3× and CD8-4×) showed improved cytotoxicity ofK562-EGFRvIII cells compared to EGFRvIII-specific CAR-T cells lackingextended stalk region (CD8-1×). Cytotoxicity exerted byEGFRvIII-specific CAR-T cells with longer CD8α derived spacers (CD8-3×and CD8-4×) was improved especially at lower E:T ratios (5:1 and 1:1)suggesting increased potency of these CAR-T cells compared to CAR-Tcells lacking stalk extension region (CD8-1×).

While preferred embodiments of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the present disclosure. It should beunderstood that various alternatives to the embodiments describedherein, or combinations of one or more of these embodiments or aspectsdescribed therein may be employed in practicing the present disclosure.It is intended that the following claims define the scope of the presentdisclosure and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

1. (canceled)
 2. The method of claim 85, wherein the stalk region isfrom about 20 to about 60 amino acids in length and the spacer regioncomprises from 1 to 5 stalk extension regions with each stalk extensionregion comprising fewer dimerization sites than the stalk region. 3-4.(canceled)
 5. The method of claim 85, wherein the stalk region isproximal to the membrane region and the stalk extension region lacks adimerization site. 6-13. (canceled)
 14. The method of claim 85, whereinthe stalk extension region comprises an amino acid sequence having atleast 80% identity with the amino acid sequence of the stalk region.15-19. (canceled)
 20. The method of claim 85, wherein the stalk regioncomprises a sequence with at least about 80% identity to a CD8alphahinge domain, a CD28 hinge domain, or a CTLA-4 hinge domain.
 21. Themethod of claim 85, the stalk region comprises an amino acid sequencehaving at least 80% identity SEO ID NO:
 3. 22-26. (canceled)
 27. Themethod of claim 85, wherein the antigen binding region binds an epitopeon CD19, BCMA, CD44, α-Folate receptor, CAIX, CD30, ROR1, CEA, EGP-2,EGP-40, HER2, HER3, Folate-binding Protein, GD2, GD3, IL-13R-a2, KDR,EDB-F, mesothelin, CD22, EGFR, Folate receptor α, a mucin, GPC3, CSPG4,HER1/HER3, HER2, CD44v6, CD44v7/v8, CD20, CD174, CD138, L1-CAM, FAP,c-MET, PSCA, CS1, CD38, IL-11Rα, EphA2, CLL-1, MAGE-A1, h5T4, PSMA,TAG-72, EGFR, CD20, EGFRvIII, CD123, and/or VEGF-R2.
 28. The method ofclaim 85, wherein the chimeric polypeptide comprises a chimeric antigenreceptor (CAR). 29-30. (canceled)
 31. The method of claim 85, whereinthe chimeric polypeptide further comprises a signaling domain from4-1BB, CD28, or a combination thereof.
 32. The method of claim 85,wherein the chimeric polypeptide further comprises a CD3-zeta signalingdomain. 33-43. (canceled)
 44. The method of claim 85, wherein the cellfurther comprises a Sleeping Beauty transposase.
 45. The method of claim44, wherein the Sleeping Beauty transposase is SB11, SB100X or SB110.46-47. (canceled)
 48. The method of claim 85, wherein the cell is ahuman T cell or NK cell. 49-68. (canceled)
 69. The method of claim 85,wherein the chimeric polypeptide comprises the amino acid sequence ofany one of SEQ ID NOs: 53-68, or a functional variant thereof having atleast 80% sequence identity therewith. 70-84. (canceled)
 85. A methodfor treating a hyperproliferative disorder, the method comprisingadministering to a subject in need thereof a cell expressing a chimericpolypeptide comprising: (a) an antigen binding region; (b) atransmembrane region; and (c) a spacer region connecting thetransmembrane and antigen binding regions, wherein the spacer regioncomprises: (i) a stalk region comprising at least one dimerization site;and (ii) a stalk extension region comprising fewer dimerization sitesthan the stalk region.
 86. The method of claim 85, wherein the antigenbinding domain binds an epitope on CD19, CD33, ROR1, mesothelin, CD22,and/or MUC-16.
 87. The method of claim 85, wherein the antigen bindingregion binds an epitope on CD19, CD33, ROR1, and/or EGFR.
 88. The methodof claim 85, wherein the antigen binding region binds an epitope onROR1.
 89. The method of claim 85, wherein the cell is administered tothe subject without undergoing propagation and activation.
 90. Themethod of claim 85, wherein the spacer region comprises an amino acidsequence having at least 80% identity with SEQ ID NO:
 5. 91. The methodof claim 85, wherein the spacer region comprises an amino acid sequencehaving the sequence of SEQ ID NO: 5.